Method for making insulin precursors and insulin precursor analogs

ABSTRACT

Novel insulin precursors and insulin precursor analogs having a mini C-peptide comprising at least one aromatic amino acid residue have an increased folding stability. The novel insulin precursors and insulin precursor analogs can be expressed in yeast in high yields and are preferably not more 15 amino acid residues in length. Also provided are polynucleotide sequences encoding the claimed precursors or precursor analogs, and vectors and cell lines containing such polynucleotide sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No. 09/736,611 filed Dec. 14, 2000 issued as U.S. Pat. No. 6,521,738, and claims priority under 35 U.S.C. 119 of Danish application no. PA 1999 01868 filed Dec. 29, 1999; Danish application no. PA 2000 00440 filed Mar. 17, 2000; U.S. application No. 60/181,443 filed Feb. 10, 2000 and U.S. application No. 60/211,441 filed Jun. 13, 2000, the contents of which are fully incorporated herein by reference.

BACKGROUND

Yeast organisms produce a number of proteins that have a function outside the cell. Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829–852).

Insulin is a polypeptide hormone secreted by β-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.

The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.

Three major methods have been used for the production of human insulin in microorganisms. Two involve Escherichia coli, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7^(th) American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp 729–739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401–5404). A third method utilizes Saccharomyces cerevisiae to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766–6770). The prior art discloses a limited number of insulin precursors which are expressed in either E. coli or Saccharomyces cerevisiae, vide U.S. Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.

Circular Dichroism (CD) is used to determine protein stability and relative stabilities of molecules. CD observed below 240 nm is due to the peptide amide chromophore and may be used to estimate protein secondary structure (Johnson (1988) Ann. Rev. Biophys. Chem. 17:145–166). The spectrum of insulin is characterized by minima at 220 and 209 nm, a negative to positive crossover near 203 nm, and a maximum at 195 nm. Upon denaturation, the negative CD in the 240–218-nm range gradually diminishes, consistent with the loss of ordered secondary structure that accompanies protein unfolding. Consequently, the folding stability of an insulin precursor may be quantitated by measuring the loss of secondary structure as a function of added denaturant, e.g., guanidinium hydrochloride (GuHCl) (see e.g., Pace (1975) CRC Crit. Rev. Biochem. 3:1–43).

SUMMARY OF THE INVENTION

The present invention features novel connecting peptides (C-peptides) which confer an increased production yield and/or increased stability in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or an insulin analog by one or more suitable, well known conversion steps.

The connecting peptides of the present invention contain at least one aromatic amino acid residue Phe, Trp, or Tyr and will generally be shorter than the natural human C peptide which, including the flanking dibasic cleavage sites, consists of 35 amino acids. Thus the novel connecting peptides will in general not be of more than 15 amino acid residues in length and preferably not more than 9 amino acid residues. Typically the novel connecting peptides will be of up to 7 or up to 5 amino acid residues and preferably not more than 4 amino acid residues.

As in the natural human insulin molecule, the connecting peptide will contain a cleavage site at its C and N termini enabling in vitro cleavage of the connecting peptide from the A and B chains. Such cleavage sites may be any convenient cleavage sites known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases; Acromobactor lyticus protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.

Alternatively, cleavage of the connecting peptide from the B chain may be enabled by cleavage at the natural Lys^(B29) amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analog. The desired B30 amino acid residue may then be added by well known in vitro, enzymatic procedures.

In one embodiment the connecting peptide will not contain two adjacent basic amino acid residues (Lys,Arg). In this embodiment, cleavage from the A-chain may be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain and the natural Lys in position B29 in the B-chain.

The connecting peptide may comprise more than one aromatic amino acid residue but preferably not more than 5. The aromatic amino acid residues may be the same or different. The connecting peptide will preferably not comprise more than 3 aromatic amino acid residues and most preferred it will only comprise a single aromatic amino acid residue.

In one embodiment of the present invention one of the aromatic amino acid residues in the connecting peptide is immediately N-terminal to the cleavage site adjacent to the A chain. Furthermore, one of the aromatic amino acid residues will preferably be positioned less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain. In one embodiment, the aromatic amino acid immediately N-terminal to the cleavage site adjacent to the A chain is less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain.

The insulin precursors or insulin precursor analogs are characterized by having a high folding stability in solution. The precursors according to the present invention will have an increased Cmid stability compared to insulin or insulin analogs, which do not comprise an aromatic amino acid residue in the connecting peptide. The Cmid stability is thus higher than about 5.5 M GuHCl, typically higher than about 6.0 M GuHCl and more typically higher than about 6.5 M GuHCl.

Accordingly, in one aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and comprising at least one aromatic amino acid residue and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one aromatic amino acid residue is immediately N-terminal to said cleavage site.

In another aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and consisting of up to 9 amino acid residues of which at least one is an aromatic amino acid residue.

In still a further aspect the present invention relates to an insulin precursor or an insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains, wherein the connecting peptide contains one aromatic amino acid residue which is less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain.

In still a further aspect the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) comprising at least one aromatic amino acid residue and being cleavable from the A and B chains Said insulin precursors or insulin precursor analogs having an increased Cmid stability relative to insulin precursor or insulin precursor analogs which do not comprise an aromatic amino acid residue a the connecting peptide.

The increased activity is determined by a variety of methods known to one of skill in the art, and described below. In one embodiment, increased stability is measured by CD determination of the concentration of guanidine hydrochloride (GuHCl) needed to achieve half-maximum unfolding of an insulin precursor molecule (Cmid).

In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1–27)-X₂-X₃-X₁-Y-A(1–21) wherein

X₁ is a peptide sequence of 1–15 amino acid residues comprising one aromatic amino acid residue immediately N-terminal to Y,

X₂ is one of Pro, Asp, Lys, or Ile at position 28 of the B chain,

X₃ is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and

Y is Lys or Arg.

In one embodiment, the total number of amino acid residues in X₁ will be from 1–10, 1–9, 1–8, 1–7, 1–6, 1–5 or 1–4 amino acid residues in length. In another specific embodiment X₁ is 1–3 amino acid residues and preferably 1–2 amino acid residues. The amino acid residues in X₁ can be any codable amino acid residue and may be the same or different with the only proviso that one is an aromatic amino acid residue immediately N-terminal to Y.

In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1–27)-X₂-X₃-X₁-Y-A(1–21) wherein

X₁ is a peptide sequence of 1–15 amino acid residues of which one is an aromatic amino acid residue which is less than 5 Å away from at least one of the amino acid residues in position B11, B12 or B26 in the B chain,

X₂ is one of Pro, Asp, Lys, or Ile at position 28 of the B chain,

X₃ is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and

Y is Lys or Arg.

In one embodiment the number of amino acid residues in X, is 1–9, 1–5 or 1–4. In another embodiment the number of amino acid residues is 1–3 or 1–2.

In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1–27)-X₂-X₃-X₁-Y-A(1–21) wherein

X₁ is a peptide sequence of 1–8 amino acid residues of which at least one is an aromatic amino acid residue,

X₂ is one of Pro, Asp, Lys, or Ile at position 28 of the B chain,

X₃ is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and

Y is Lys or Arg.

The total number of amino acid residues in X₁ will be from 1–7, 1–6, 1–5 or 1–4 amino acid residues. In a more specific embodiment X₁ is 1–3 amino acid residues and preferably 1–2 amino acid residues. The amino acid residues in X₁ can be any codable amino acid residue and may be the same or different with the only proviso that at least one amino acid residue in X₁ is an aromatic amino acid residue.

In the above formulas X₁ may comprise up to 5 aromatic amino acid residues which may be the same or different. In a specific embodiment, X₁ comprises up to 3 aromatic amino acid residues which may be the same or different and X₁ will preferably contain only one aromatic amino acid residue. The aromatic amino acid residues are Trp, Phe or Tyr, preferably Phe or Trp.

In one embodiment, X₂ is Asp and X₃ is Lys. This embodiment encompasses the insulin precursor analogs containing an Asp in position B28 of the B chain (termed hereinafter “Asp^(B28)IP”). In another embodiment X₂ is Lys and X₃ is Pro. In a further embodiment the sequence X₁-Y is selected from the group of:

(a) Met-Trp-Lys; (b) Ala-Trp-Lys; (c) Val-Trp-Lys; (d) Ile-Trp-Lys; (e) Leu-Trp-Lys; (f) Glu-Glu-Phe-Lys (SEQ ID NO:15); (g) Glu-Phe-Lys; (h) Glu-Trp-Lys; (i) Ser-Trp-Lys; (j) Thr-Trp-Lys; (k) Arg-Trp-Lys; (l) Glu-Met-Trp-Lys (SEQ ID NO:1); (m) Gln-Met-Trp-Lys (SEQ ID NO:2); and (n) Asp-Trp-Lys.

In another embodiment X₂ is Pro, X₃ is Lys and X₁ is 1–2 amino acid residues of which one is Trp or Phe.

In another embodiment X₂ is Lys, X₃ is Pro-Thr and X₁ consists of up to 15 amino acid residues of which one is Trp, Tyr or Phe. In this embodiment X₁ will contain a cleavage site at the C-terminal end, e.g a mono basic or dibasic (Lys, Arg) cleavage site.

The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin precursor analogs. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and host cell containing such polynucleotide sequences or vectors.

In another aspect, the invention relates to a process for producing the insulin precursors or insulin precursor analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention under suitable conditions for expression of said precursor or precursor analog; and (ii) isolating the precursor or precursor analog from the culture medium.

In still a further aspect, the invention relates to a process for producing insulin or insulin analogs in a host cell said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin precursor analogs of the invention; (ii) isolating the precursor or precursor analog from the culture medium and (iii) converting the precursor or precursor analog into insulin or an insulin analog by in vitro enzymatic conversion.

In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:3)-IP(AlaAlaLys) fusion protein.

FIG. 2 is the DNA sequence and inferred amino acid sequence of the encoded fusion protein (α-factor-leader-EEAEAEAPK(SEQ ID NO:4)-Asp^(B28)IP portion of pAK1150 (SEQ ID NO: 5 and 6) used as PCR template.

FIG. 3 is the DNA sequence encoding α-factor leader-Asp^(B28)IP(GluTrpLys) fusion protein with a synthetic mini C-peptide GluTrpLys generated by randomized optimization (SEQ ID NO:7 and 8). The mini C-peptide (EWK) is indicated by underlining.

FIG. 4 shows folding stability of the insulin analog Asp^(B28)IP(MetTrpLys) relative to Asp^(B28)IP.

FIG. 5 shows the solution structures of Asp^(B28)IP(MetTrpLys) as backbone lines of ensemble of 20 converged structures.

FIG. 6 shows a ribbon presentation of Asp^(B28)IP(MetTrpLys). The figure is produced using MOLSCRIPT (Kraulis (1991) J. Appl. Crystallog. 24:946–950). Amino acid residue annotations are derived as follows: B1–B29 (B chain) are numbered 1–29, residues C1–C3 (connecting peptide) are numbered 30–32, and residues A1–A21 (A chain) are numbered 33–53.

FIG. 7 is the ID proton NMR spectrum for Asp^(B28)IP(MetTrpLys) recorded at 27° C. at 1.0 mM concentration in 10%/90% D₂O/H₂O with 10 mM phosphate buffer at pH 8.0.

FIG. 8 is the DNA and inferred amino acid sequence of the expression cassette expressing the YAP3-TA39-EEGEPK(SEQ ID NO:17)-Asp^(B28)IP fusion protein with a synthetic mini C-peptide GluTrpLys (SEQ ID NO:9 and 10) and

FIG. 9 is the DNA and inferred amino acid sequence of the expression cassette expressing plasmid expressing the YAP3-TA57-EEGEPK(SEQ ID NO:17)-Asp^(B28)IP fusion protein with a synthetic mini C-peptide GluTrpLys (SEQ ID NO:11 and 12).

DETAILED DESCRIPTION

Abbreviations and Nomenclature.

By “connecting peptide” or “C-peptide” is meant the connection moiety “C” of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain. A “mini C-peptide” or “connecting peptide” such as those described herein, connect B29 or B30 to A1, and differ in sequence and length from that of the natural C-peptide.

By “IP” is meant a single-chain insulin precursor in which a desB30 chain is linked to the A chain of insulin via a connecting peptide. The single-chain insulin precursor will contain correctly positioned disulphide bridges (three) as in human insulin.

With “desB30” or “B(1–29)” is meant a natural insulin B chain lacking the B30 amino acid residue, “A(1–21)” means the natural insulin A chain, “B(1–27)” means the natural B chain lacking the B28, B29, and B30 amino acid residues; “Asp^(B28)IP” means a single-chain insulin precursor with aspartic acid at position 28 of the B-chain and no C-peptide (B29 is linked to A1). The mini C-peptide and its amino acid sequence is indicated in the three letter amino acid code in parenthesis following the IP; Thus “Asp^(B28)IP(MetTrpLys)” means a single-chain insulin precursor with aspartic acid at position 28 of the B-chain and a mini C-peptide with the sequence Met-Trp-Lys connecting B29 to A1.

By “insulin precursor” is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin.

By “insulin precursor analog” is meant an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogs are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue. In one embodiment, the instant invention comprises analog molecules having position 28 of the B chain altered relative to the natural human insulin molecule. In this embodiment, position 28 is modified from the natural Pro residue to one of Asp, Lys, or Ile. In a preferred embodiment, the natural Pro residue at position B28 is modified to an Asp residue. In another embodiment Lys at position B29 is modified to Pro; Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys. Further examples of insulin precursor analogs are des(B30) human insulin, insulin analogs wherein Phe^(B1) has been deleted; insulin analogs wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogs wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1.

The term “immediately N-terminal to” is meant to illustrate the situation where an amino acid residue or a peptide sequence is directly linked at its C-terminal end to the N-terminal end of another amino acid residue or amino acid sequence by means of a peptide bond.

In the present context, the term “functional analog of insulin” and the like, is meant to indicate a polypeptide with a similar biological action as the native human insulin protein.

By a distance shorter than 5 Å between two amino acid residues is meant the shortest inter-atomic distance less than 5 Å between any atom in the first amino acid and any atom in the second amino acid. Atomic distances are measured from three-dimensional structures of the molecule determined either by NMR (Wüthrich, K., 1986, NMR of Proteins and Nucleic Acids, Wiley, New York) or by X-ray crystallography (Drenth, J., 1994, Principles of Protein X-ray crystallography, Springer Verlag Berlin). A distance from one amino acid to another is measured as the shortest inter-atomic distance between any atom in the first amino acid and any atom in the second amino acid if not stated differently.

The present invention features novel mini C-peptides connecting position 29 of the insulin B chain and position 1 of the insulin A chain which significantly increased production yields in a yeast host cell. By the term “significantly increased production,” “increased fermentation yield,” and the like, is meant an increase in secreted amount of the insulin precursor molecule or insulin precursor analog molecule present in the culture supernatant compared to the yield of an insulin precursor or insulin precursor analog with no aromatic amino acid residue in the mini C peptide. An “increased” fermentation yield is an absolute number larger than the control; preferably, the increase is 50% or more larger than the control (Asp^(B28)IP) level; even more preferably, the increase is 100% or more larger than control levels.

By the term “increase in stability” is meant, for example, an increased value of Cmid in solution relative to that obtained for an insulin analog precursor (e.g., Asp^(B28)IP) without an aromatic amino acid residue in the mini C-peptide. By the term “Cmid” is meant the concentration of GuHCl necessary to unfold one-half of the protein population in an assay measuring the far-UV circular dichroism of the insulin molecule as a function of increasing concentrations of denaturant.

“POT” is the Schizosaccharomyces pombe triose phosphate isomerase gene, and “TPI1” is the S. cerevisiae triose phosphate isomerase gene.

By a “leader” is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide.

The term “signal peptide” is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein. The function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. A number of signal peptides which may be used with the DNA construct of the invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al. (1990) YEAST 6:127–137 and U.S. Pat. No. 5,726,038) and the α-factor signal of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathern et al., eds., pp 143–180, Cold Spring Harbor Laboratory, NY and U.S. Pat. No. 4,870,00.

The term “pro-peptide” means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The pro-peptide may be the yeast α-factor pro-peptide, vide U.S. Pat. Nos. 4,546,082 and 4,870,008. Alternatively, the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in U.S. Pat. Nos. 5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro-peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence or any functional analog thereof.

The polynucleotide sequence of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859–1869, or the method described by Matthes et al. (1984) EMBO Journal 3:801–805. According to the phosphoamidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR).

The polynucleotide sequence of the invention may also be of mixed genomic, cDNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides.

The invention encompasses a vector which is capable of replicating in the selected microorganism or host cell and which carries a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

In a preferred embodiment, the recombinant expression vector is capable of replicating in yeast Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 μm replication genes REP 1–3 and origin of replication.

The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyl-transferase), pyrG (orotidine-5′-phosphate decarboxylase) and trpC (anthranilate synthase. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A preferred selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40:125–130).

In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intra-cellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene (penP). Examples of suitable promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1, TPI, ADH or PGK promoters.

The polynucleotide construct of the invention will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1:419–434).

The procedures used to ligate the polynucleotide sequence of the invention, the promoter and the terminator, respectively, and to insert them into suitable yeast vectors containing the information necessary for yeast replication, are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence encoding the insulin precursors or insulin precursor analogs of the invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, mini C-peptide, A and B chains) followed by ligation.

The present invention also relates to recombinant host cells, comprising a polynucleotide sequence encoding the insulin precursors or the insulin precursor analogs of the invention. A vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect, plant, or fungal cells. In a preferred embodiment, the host cell is a yeast cell. The yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the insulin precursor and insulin precursor analogs of the invention. Examples of suitable yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans.

The transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted insulin precursor or insulin precursor analogs of the invention, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin precursor or insulin precursor analog by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.

The insulin precursors and insulin precursor analogs of the invention may be expressed with an N-terminal amino acid residue extension, as described in U.S. Pat. No. 5,395,922, and European Patent No. 765,395A, both of which patents are herein specifically incorporated by reference. The extension is found to be stably attached to the insulin precursor or insulin precursor analogs of the invention during fermentation, protecting the N-terminal end of the insulin precursor or insulin precursor analog against the proteolytic activity of yeast proteases such as DPAP. The presence of an N-terminal extension on the insulin precursor or insulin precursor analog may also serve as a protection of the N-terminal amino group during chemical processing of the protein, i.e. it may serve as a substitute for a BOC (t-butyl-oxycarbonyl) or similar protecting group. The N-terminal extension may be removed from the recovered insulin precursor or insulin precursor analog by means of a proteolytic enzyme which is specific for a basic amino acid (e.g., Lys) so that the terminal extension is cleaved off at the Lys residue. Examples of such proteolytic enzymes are trypsin or Achromobacter lyticus protease.

After secretion to the culture medium and recovery, the insulin precursor or insulin precursor analogs of the invention will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and the mini C-peptide to give insulin or the desired insulin analog. Such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin or insulin analog into insulin or the insulin analog by basic or acid hydrolysis as described in U.S. Pat. Nos. 4,343,898 or 4,916,212 or Research Disclosure, September 1994/487 the disclosures of which are incorporated by reference hereinto.

As described below, insulin precursors or insulin precursor analogs with synthetic C-peptides were constructed featuring at least one aromatic amino acid (Example 1). Saccharomyces cerevisiae expression plasmids containing a polynucleotide sequence encoding the claimed insulin precursors or insulin precursor analogs were constructed by PCR and used to transform a S. cerevisiae host cell. The amount of expressed product, e.g. an insulin analog was measured as a percentage of the relevant control level, e.g. amount of expressed Asp^(B28)IP lacking mini C-peptide (Table 1) and Asp^(B28)IP(AlaAlaLys) with a mini C-peptide without an aromatic amino acid residue (Table 2). The novel C-peptides of the invention gave increased yields by up to 7-fold levels.

The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein.

EXAMPLES

General Procedures

All expressions plasmids are of the C-POT type, similar to those described in EP 171,142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae. The plasmids also contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-XbaI fragment encoding the fusion protein of the leader and the insulin precursor product. In order to express different fusion proteins, the EcoRI-XbaI fragment of pKFN1003 is simply replaced by an EcoRI-XbaI fragment encoding the leader-insulin precursor-fusion of interest. Such EcoRI-XbaI fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.

Yeast transformants were prepared by transformation of the host strain S. cerevisiae strain MT663 (MATa/MATα pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir⁺). The yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278.

MT663 was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na₂EDTA pH=8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30° C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na₂EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym®234. The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (Tris=Tris(hydroxymethyl)aminomethane) pH=7.5) and resuspended in 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl₂, 10 mM Tris HCl, pH=7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium.

S. cerevisiae strain MT663 transformed with expression plasmids was grown in YPD for 72 h at 30° C. Quantitation of the insulin-precursor yield in the culture supernatants was performed by reverse-phase HPLC analysis with human insulin as an external standard (Snel & Damgaard (1988) Proinsulin heterogenity in pigs. Horm. Metabol. Res. 20:476–488).

Example 1 Construction of Synthetic C-peptides with Aromatic Amino Acid(s)

Synthetic genes encoding fusion proteins, consisting of AsP^(B28)IP associated with a leader sequence consisting of a pre-peptide (signal peptide) and a pro-peptide, were constructed using PCR under standard conditions (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) and E.H.F. polymerase (Boehringer Mannheim GmbH, Sandhoefer Strasse 116, Mannheim, Germany). The resulting DNA fragments were isolated and digested with endonucleases and purified using the Gene Clean kit (Bio101 Inc., La Jolla, Calif., USA). Standard methods were used for DNA ligation and transformation of E. coli cells were performed by the CaCl₂ method (Sambrook et al. (1989) supra). Plasmids were purified from transformed E. coli cells using QIAGEN columns (QIAGEN, Hilden, Germany). Nucleotide sequences were determined using the ALF Pharmacia Biotech DNA sequencing system with purified double-stranded plasmid DNA as template. Oligonucleotide primers for PCR were obtained from DNA technology (Arhus, Denmark).

Secretory expression of the Asp^(B28)IP in S. cerevisiae was performed using the S. cerevisiae strain MT663 and the 2 μm based yeast expression vector CPOT (see FIG. 1) as described in Thim, L. et al. (1986) Proc. Natl. Acad. Sci. USA 83:6766–6770. The yeast expression vector contains the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for plasmid selection and stabilization in S. cerevisiae. Furthermore, the S. cerevisiae triose phosphate isomerase gene (TPI1) promoter and terminator are used for transcription initiation and termination of the recombinant gene encoding the leader-Asp^(B28)IP fusion protein.

Secretion of the AsP^(B28)IP was facilitated by the α-factor leader, although a variety of known yeast leader sequences may be used.

As shown in FIG. 1, the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:3)-IP fusion protein was constructed based on the S. cerevisiae-E. coli shuttle POT plasmid (U.S. Pat. No. 5,871,957). In FIG. 1 L-IP indicates the fusion protein expression cassette encoding the leader-IP fusion protein; TPI-PROMOTER is the S. cerevisiae TPI1 promoter, TPI-TERMINATOR is the S. cerevisiae TPI1 terminator; TPI-POMBE indicates the S. pombe POT gene used for selection in S. cerevisiae; ORIGIN indicates a S. cerevisiae origin of replication derived from the 2 μm plasmid; AMP-R indicates the β-lactamase gene conferring resistance toward ampicillin, facilitating selection in E. coli; and ORIGIN-PBR322 indicates an E. coli origin of replication.

DNA encoding a number of fusions proteins of leader sequences and AsP^(B28)IP with different mini C-peptides was generated by PCR using appropriate oligonucleotides as primers, as described below. Standard methods were used to subclone DNA fragments encoding the leader-Asp^(B28)IP fusion proteins into the CPOT expression vector in the following configuration: leader-Lys-Arg-spacer-Asp^(B28)IP, where Lys-Arg is a potential dibasic endoprotease processing site and spacer is an N-terminal extension. To optimize processing of the fusion protein by the S. cerevisiae Kex2 endoprotease, DNA encoding a spacer peptide (N-terminal extension), e.g. EEAEAEAPK (SEQ ID NO:4), was inserted between the DNA encoding the leader and the AsP^(B28)IP (Kjeldsen et al. (1996) Gene 170, 107–112.). However, the present of the spacer peptide is not mandatory. The mature Asp^(B28)IP was secreted as a single-chain N-terminally extended insulin precursor with a mini C-peptide, connecting Lys^(B29) and Gly^(A1). After purification of the Asp^(B28)IP and proteolytic removal of the N-terminal extension and the mini C-peptide, the amino acid Thr^(B30) can be added to Lys^(B29) by enzyme-mediated transpeptidation, to generate Asp^(B28) human insulin (Markussen, et al. (1987) in “Peptides 1986” (Theodoropoulos, D., Ed.), pp. 189–194, Walter de Gruyter & Co., Berlin.).

Development of synthetic mini C-peptides was performed by randomization of one or more codon(s) encoding the amino acids in the mini C-peptide. All synthetic mini C-peptides feature an enzymatic processing site (Lys) at the C-terminus which allows enzymatic removal of the synthetic mini C-peptide (U.S. Pat. No. 4,916,212, herein specifically incorporated by reference). Randomization was performed using doped oligonucleotides which introduced codon(s) variations at one or more positions of the synthetic mini C-peptides. Typically one of the two primers (oligonucleotides) used for PCR was doped. An example of an oligonucleotides pair used for PCR generation of leader-Asp^(B28)IP with randomized synthetic mini C-peptides used to generated synthetic mini C-peptides with the general formula: Xaa-Trp-Lys (XWK) are as follows:

Primer A: 5′-TAAATCTATAACTACAAAAAACACATA-3′ and (SEQ ID NO:13) Primer B: 3′-CCAAAGAAGATGTGACTGTTCNNMACCTTCCCATAGCAACTTGTTACAAC- (SEQ ID NO:14) ATGAAGATAGACAAGAAACATGGTTAACCTTTTGATGACATTGATCAGATCTTTGA-TTC-5′, where N is A, C, G, or T and M is C or A.

Polymerase chain reaction. PCR was typically performed as indicated below: 5 μl Primer A (20 pmol), 5 μl Primer B (20 pmol), 10 μl 10×PCR buffer, 8 μl dNTP mix, 0.75 μl E.H.F. enzyme, 1 μl pAK1150 plasmid as template (approximately 0.2 μg DNA) and 70.25 μl distilled water.

Typically between 10 and 15 cycles were performed, one cycle typically was 94° C. for 45 sec.; 55° C. for 1 min; 72° C. for 1.5 min. The PCR mixture was subsequently loaded onto a 2% agarose gel and electrophoresis was performed using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated by the Gene Clean kit.

FIG. 2 shows the sequence of pAK1150 DNA used as template for PCR and inferred amino acids of the encoded fusion protein (α-factor-leader-(EEAEAEAPK)(SEQ ID NO:4)-Asp^(B28)IP of pAK1150 (SEQ ID NO:5 and 6). The pAK1150 plasmid is similar to pAK721 shown in FIG. 1. The α-factor-leader's C-terminus was modified to introduced a Nco I restriction endonuclease site, which changes the inferred amino acid sequences from SerLeuAsp to SerMetAla. Moreover, the encoded Asp^(B28)IP does not feature a mini C-peptide but Lys^(B29) is directly connected to Gly^(A1).

The purified PCR DNA fragment was dissolved in water and restriction endonucleases buffer and digested with suitable restriction endonucleases (e.g. Bgl II and Xba I) according to standard techniques. The BglII-XbaI DNA fragments were subjected to agarose electrophoresis and purified using The Gene Clean Kit.

The expression plasmid pAK1150 or a similar plasmid of the CPOT type (see FIG. 1) was digested with the restriction endonucleases Bgl II and Xba I and the vector fragment of 10765 nucleotide basepairs isolated using The Gene Clean Kit.

The two digested and isolated DNA fragments (the vector fragment and the PCR fragment) were ligated together using T4 DNA ligase and standard conditions. The ligation mix was subsequently transformed into a competent E. coli strain (R−, M+) followed by selection with ampicillin resistance. Plasmids from the resulting E. coli's were isolated using QIAGEN columns.

The plasmids were subsequently used for transformation of a suitable S. cerevisiae host strain, e.g., MT663 (MATa/MATα pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir⁺). Individual transformed S. cerevisiae clones were grown in liquid culture, and the quantity of Asp^(B28)IP secreted to the culture supernatants were determined by RP-HPLC. The DNA sequence encoding the synthetic mini C-peptide of the expression plasmids from S. cerevisiae clones secreting increased quantity of the Asp^(B28)IP were then determined. Subsequently, the identified synthetic mini C-peptide sequence might be subjected to another round of randomization optimization.

An example on a DNA sequence encoding a leader-Asp^(B28)IP(GluTrpLys) fusion protein featuring a synthetic mini C-peptide (GluTrpLys) resulting from the randomized optimization process described is shown in FIG. 3 (SEQ ID NO:7 and 8).

Table 1 and 2 show the insulin precursors analogs generated by the above method and production yield expressed as a percent of control. Fermentation was conducted at 30° C. for 72 h in 5 ml YPD. Yield of the insulin precursor was determined by RP-HPLC of the culture supernatant, and is expressed relative to the yield of a control strain expressing either a leader-Asp^(B28)IP fusion protein in which the B29 residue is linked to the A1 residue by a peptide bond; or leader-Asp^(B28)IP fusion protein in which the B29 residue is linked to the A1 residue by a mini C-peptide, respectively. In the tables, “α*” indicates an α-factor leader in which the C-terminus up to the LysArg has been modified from “SLD (SerLeuAsp)” to “SMA (SerMetAla)” and “ex4” is an N-terminal extension with the amino acid sequence EEAEAEAPK(SEQ ID NO:4). YAP3 is the YAP3 signal sequence TA39 is a synthetic pro-sequence QPIDDTESNTTSVNLMADDTESRFATNTTLAGGLDVVNLISMAKR (SEQ ID NO:16). The sequence EEGEPK (SEQ ID NO:17) is an N-terminal extension to the B-chain of the insulin analogue. TA57 is a synthetic pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR

TABLE 1 Leader-N- terminal ex- tension Precursor mini C-peptide Yield* SEQ ID NO: α*-ex4 Asp^(B28) IP None 100 (control) α*-ex4 Asp^(B28) IP MetTrpLys 378 α*-ex4 Asp^(B28) IP AlaTrpLys 270 α*-ex4 Asp^(B28) IP ValTrpLys 284 α*-ex4 Asp^(B28) IP IleTrpLys 330 α*-ex4 Asp^(B28) IP LeuTrpLys 336 α*-ex4 Asp^(B28) IP LysTrpLys 288 α*-ex4 Asp^(B28) IP GluGluPheLys 272 SEQ ID NO: 15 α*-ex4 Asp^(B28) IP GluPheLys 379 α*-ex4 Asp^(B28) IP GluTrpLys 374 α*-ex4 Asp^(B28) IP SerTrpLys 226 α*-ex4 Asp^(B28) IP ThrTrpLys 270 α*-ex4 Asp^(B28) IP ArgTrpLys 227 α*-ex4 Asp^(B28) IP GluMetTrpLys 212 SEQ ID NO: 1 α*-ex4 Asp^(B28) IP GlnMetTrpLys 239 SEQ ID NO: 2

TABLE 2 Leader-N- terminal extension Precursor Mini C-peptide Yield* SEQ ID NO: α*-ex4 Asp^(B28)IP AlaAlaLys 100 (control) α*-ex4 Asp^(B28)IP GluTrpLys 626 α*-ex4 Asp^(B28)IP GluTyrLys 466 α*-ex4 Asp^(B28)IP GluPheLys 444 α*-ex4 Asp^(B28)IP AspTrpLys 460 YAP3-TA57- Asp^(B28)IP GluTrpLys 767 (SEQ ID NO: 17) EEGEPK YAP3-TA39- Asp^(B28)IP MetTrpLys 687 (SEQ ID NO: 17) EEGEPK

Example 2 Structure Determination of Asp^(B28)IP(MetTrpLys) in Aqueous Solution by NMR Spectroscopy

NMR spectroscopy. Samples for NMR were prepared by dissolving the lyophilized protein powder in 10/90 D₂O/H₂O with a 10 mM phosphate buffer and adjusting the pH as desired by addition of small volumes of 1 M DCl or NaOD. All pH meter readings are without correction for isotope effects. Samples of Asp^(B28)IP(MetTrpLys) for NMR were prepared at concentrations ranging from 25 μM to 1 mM at pH 8.0. Two-dimensional ¹H-¹H NMR spectra of 1 mM samples, DQF-COSY (Piantini et al. (1982) J. Am. Chem. Soc. 104:6800–6801, Rance et al. (1983) Biochem. Biophys. Res. Commun. 117:479–485), TOCSY (Braunschweiler et al. (1983) J. Magn. Reson. 53:521–528, Bax et al. (1985) J. Magn. Reson. 65:355–360) and NOESY (Jeener et al. (1979) J. Chem. Phys. 71:4546–4553) were recorded at 600 MHz on a Varian Unity Inova NMR spectrometer equipped with a ¹H/¹³C/¹⁵N triple resonance probe with a self-shielded triple-axis gradient coil using standard pulse sequences from the Varian user library. The operating temperature was set to 27° C. For each phase sensitive two-dimensional NMR spectrum 512 t₁ increments were acquired each with 2048 or 4096 real data points according to the TPPI-States method (Marion et al. (1989) J. Magn. Reson. 85:393–399). Spectral widths of 6983 Hz in both dimensions were used, with the carrier placed exactly on the water resonance which was attenuated by using either saturation between scans for 1.5 seconds or selective excitation by a gradient-tailored excitation pulse sequence (WATERGATE, Piotto et al. (1992) J. Biomol. NMR 2:661–665). DQFCOSY spectra were recorded using a gradient enhanced version applying magic-angle gradients (Mattiello et al. (1996) J. Am. Chem. Soc. 118:3253–3261). For TOCSY spectra mixing times between 30 and 80 ms were used and for NOESY mixing times between 50 and 200 ms.

The processing of the two-dimensional NMR spectra was performed using the software package Xwinnmr (version 2.5, NMR processing software from Bruker Analytische Messtechnik GmbH, D-76275 Ettlingen, Germany). Each dimension was processed with shifted sine-bell apodization and zero-filling performed once in each dimension. Baseline corrections were applied if necessary using Xwinnmr standard procedures.

The spectral assignment, cross peak integration, sequence specific assignment, stereo specific assignment, and all other bookkeeping were performed using the program PRONTO (PRONTO Software Development and Distribution, Copenhagen Denmark) (Kjær et al. (1991) NATO ASI Series (Hoch, J. C., Redfield C., & Poulsen, F. M., Eds.) Plenum, New York). Chemical shifts are measured in ppm and the water resonance set to 4.75 ppm.

Structure calculations. Distance restraints for the subsequent structure calculation were obtained from integrated NOESY cross peaks classified as either weak, medium or strong corresponding to upper distance restraints of 5.5, 3.3, and 2.7 Å, respectively. For distance restraints involving methyl groups, an additional 0.5 Å was added to the upper limit (Wagner et al. (1985) J. Mol. Biol. 196:611–639). Structure calculations were performed using the hybrid method combining distance geometry (Crippen et al. (1988) Distance Geometry and Molecular Conformation, Research Studies Press, Taunton, Somerset, England; Kuszewski et al. (1992) J. Biomol NMR 2:33–56) and simulated annealing based on the ideas of Nilges et al. (1988) FEBS Lett. 229:317–324 using X-PLOR 3.0 (Brünger (1992) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR, Yale University Press, New Haven) according to the examples given by the X-PLOR manual (dg_sub_embed.inp, dgsa.inp, refine.inp). Residue numbers are derived from standard insulin residue numbering, residues in the B-chain are numbered B1–B29, residues in the C-peptide (e.g. MetTrpLys) are numbered C1–C3 and residues in the A-chain are numbered A1–A21.

Spectral assignment of the NMR spectra followed for most resonances the standard sequential assignment procedure described by Wüthrich (1986 NMR of Proteins and Nucleic Acids, Wiley, New York). The standard assignment procedure fails when the amid proton of a particular amino acid residue exchanges to rapidly with protons in the water. At pH 8.0 this occurs for several amino acid residues, however, comparison with earlier mutant insulin NMR spectral assignments and identification of neighbouring (in space) amino acid residues through NOEs allow an almost total spectral assignment. Analysis of the NOESY spectra showed that several amino acid residues had a NOE network to the surrounding residues similar to what has previously been determined for other insulin molecules, i.e., human insulin His^(B16) mutant (Ludvigsen et al. (1994) Biochemistry 33:7998–8006) and these similar connections are found for residues B1–B10, B13–B14, B17–B24 and A4–A21. Additionally the dihedral angle restraints for the above listed residues were adopted from those used previously (Ludvigsen et al. (1994) supra).

Several amino acids in particular B27–B29, C1–C3, A1–A3 have cross peaks patterns which are consistent with peptide chains that are less well ordered than commonly well-defined secondary structural elements. Thus additional NOEs were converted into distance restraints without any further classification than upper limits of 5.5 Å or 6.0 Å if a methyl group were included. An ensemble of 20 converged structures (FIG. 5) was calculated and the relevant parameters listed in Table 3 for the converged structures. Each NOE here identical to a distance restraint is only counted once even though it might occur several times in the NOESY spectrum. Ramachandran plot quality assessment is standard quality parameters to evaluate local geometry quality. In general the described quality parameters are comparable to 2.5 Å resolution of X-ray based protein structures (Laskowski et al. (1996) J. Biolmol. NMR 8:477–486).

TABLE 3 Structurea quality assessment Asp^(B28)IP(MetTrpLys) Number of NOEs Total 742 Intra 319 short range (within 5 270 residue positions away but not intra NOEs) long range (more than 153 5 residue positions away) Violations of NOEs >0.4 Å (average for 20  0 structures) RMS of NOE violations 0.013(±0.002) Å RMS of dihedral angle restraints 0.30(±0.08)° Deviations from ideal geometry Impropers 0.28(±0.02)° Angles 0.38(±0.02)° Bonds 0.0031(±0.0002) Å Ramachandran Plot (Las- Favoured regions  77.2% kowski 1996) additional allowed  19.8% regions generously allowed  2.6% regions disallowed regions  0.4% Description of the Calculated Structure.

A representative structure most resembling the average of the ensemble is displayed in FIG. 6. Asp^(B28)IP(MetTrpLys) is structurally similar to the native insulin structure for regions comprising residues B1–B10, B14–B23, A4–A21. The differences are mostly pronounced for regions in the vicinity of the connecting peptide in positions B26–B29, C1–C3, A1–A3 and less pronounced for residues B11–B13. The structure of Asp^(B28)IP(MetTrpLys) near the C-peptide is strikingly different from the native like structure (Ludvigsen (1994) supra). The methionine and tryptophan side-chains in Asp^(B28)IP(MetTrpLys) opens the traditional insulin core structure by moving on one side the side-chains of particular Tyr^(B26) and Phe^(B25) away and leaving the otherwise usual neighboring hydrophobic patch comprised by the side-chains of Leu^(B11), Val^(B12), Ile^(A2) and Tyr^(A19) intact. This pocket created by moving Phe^(B25), Tyr^(B26) and the peptide chain comprised by residues B25 to B29 is apparently well suited to accommodate the packing of the side-chains Met^(C1) and TrP^(C2) from the C-peptide. Several NOEs from these two side-chains to structurally neighboring residues verify this very new arrangement of side-chains not previously observed in any insulin structure. Met^(C1) is placed in a pocket composed by the residues Leu^(B15), Phe^(B24), Tyr^(B26), Trp^(C1), Ile^(A2) and Tyr^(A19) which all have NOEs to Met^(C1). Trp^(C2) has an even more extensive NOE network, but due to fast exchange of the indole amid proton only four resonances belonging to the aromatic ring system of Trp^(C2) can be assigned. Despite this 21 inter-residue NOEs between Trp^(C2) and its neighbors spanned by Leu^(B11), Val^(B12), Leu^(B15), Tyr^(B26), Met^(C1) and Ile^(A2) have been observed in the NOESY spectrum of Asp^(B28)IP(Met Trp Lys).

The presence of a tryptophan side-chain in the pocket also has extensive impact on the chemical shifts observed in the spectra of Asp^(B28)IP(Met Trp Lys). Under the conditions used for NMR the spectra of Asp^(B28)IP(Met Trp Lys) are influenced by some degree of self-association (FIG. 7) but the exchange between monomer and dimer is on the timescale of NMR only observed as an average between the two states. Between concentrations of 25 μM and 0.2 mM the degree of self-association does not change as seen by NMR.

Table 4 shows chemical shifts of Asp^(B28)IP(MetTrpLys) at 27° Celcius obtained at 600 MHz, pH 8 in 10%/90% D₂O/H₂O with 10 mM phosphate buffer. Chemical shifts are referenced by setting the residual water signal to 4.75 ppm. N/A means no assignment. Asp^(B28)IP(MetTrpLys) assignments (1–29=B1–B29; 30–32=C1–C3 and 33–53=A1–A21) and Table 5 provides the atomic coordinates of Asp^(B28)IP(MetTrpLys) in PDB format.

TABLE 4 NMR spectral assignments for Asp^(B28)IP(MetTrpLys) Spin system HN HA Other: Phe-1 4.52 HB#a: 2.976, HB#b: 3.040, HD#: 7.104, HE#: 7.214, HZ: 7.177 Val-2 N/A N/A HB: N/A, HG#a: N/A, HG#b: N/A Asn-3 N/A HB#a: N/A, HB#b: N/A, HD2#a: N/A, HD2#b: N/A Glu-4 N/A HB#a: N/A, HB#b: N/A His-5 4.30 HB#a: 3.311, HB#b: 2.992, HD2: 6.850, HE1: 7.605 Leu-6 4.47 HB#a: 1.635, HB#b: 0.807, HG: 1.506, HD#a: 0.753, HD#b: 0.675 Cys-7 8.30 4.83 HB#a: 2.901, HB#b: 3.173 Gly-8 N/A, N/A Ser-9 N/A HB#: N/A His-10 N/A 4.41 HB#a: 3.140, HB#b: 3.351, HD2: 7.112, HE1: 7.729 Leu-11 N/A 3.93 HB#a: N/A, HB#b: 1.143, HG: 1.257, HD#a: 0.375, HD#b: 0.559 Val-12 7.25 3.37 HB: 2.000, HG#a: 0.849, HG#b: N/A Glu-13 N/A N/A HB#a: N/A, HG#a: N/A, HG#b: N/A Ala-14 7.71 3.98 HB#: 1.307 Leu-15 7.83 3.74 HB#a: 0.671, HB#b: 1.254, HG: 1.157, HD#a: N/A, HD#b: 0.295 Tyr-16 8.15 4.31 HB#a: 3.115, HD#: 7.204, HE#: 6.734 Leu-17 7.99 4.05 HB#a: 2.004, HB#b: 1.849, HG: 1.732, HD#a: 0.900, HD#b: 0.879 Val-18 8.49 3.71 HB: 1.996, HG#a: 0.994, HG#b: 0.834 Cys-19 8.57 4.81 HB#a: 2.820, HB#b: 3.250 Gly-20 7.75 3.96, N/A Arg-22 N/A N/A HB#a: N/A, HB#b: N/A, HG#a: N/A, HG#b: N/A, HD#a: N/A, HD#b: N/A Gly-23 7.12 4.06, 3.76 Phe-24 7.56 4.99 HB#a: 2.971, HB#b: 3.206, HD#: 6.746, HE#: 6.877, HZ: N/A Phe-25 N/A 4.78 HB#a: 3.051, HB#b: N/A, HD#: 7.172, HE#: 7.249 Tyr-26 N/A 4.63 HB#a: 2.770, HB#b: N/A, HD#: 6.694, HE#: 6.291 Thr-27 N/A N/A HB: N/A, HG2#: N/A Asp-28 N/A N/A HB#a: N/A, HB#b: N/A Lys-29 Met-30 7.84 4.18 HB#: 2.627, HG#: 2.904, HE#: 2.110 Trp-31 4.19 HB#a: 3.182, HE3: 7.037, HH2: 6.763, HZ2: 7.147, HZ3: 6.437 Lys-32 Gly-33 N/A, N/A Ile-34 7.98 3.53 HB: 0.953, HG1#a: 0.382, HG1#b: 0.562, HG2#: 0.234, HD#: 0.029 Val-35 7.75 N/A, 4.00 HB: N/A, HB: 1.925, HG#a: N/A, HG#b: N/A, HG#b: 0.807 Glu-36 N/A N/A HB#a: N/A, HG#a: N/A Gln-37 7.73 3.96 HB#: 2.033, HG#: 2.313 Cys-38 8.24 4.89 HB#a: 3.103, HB#b: 2.700 Cys-39 N/A N/A HB#a: N/A, HB#b: N/A Thr-40 3.95 HB: 4.411, HG2#: 1.167 Ser-41 6.97 4.59 HB#a: 3.698, HB#b: 3.867 Ile-42 7.59 4.32 HB: 1.399, HG1#a: N/A, HG2#: 0.573, HD#: 0.389 Cys-43 N/A N/A HB#a: N/A Ser-44 N/A N/A HB#a: N/A, HB#b: N/A Leu-45 3.86 HB#a: 1.412, HB#b: 1.489, HG: 1.565, HD#a: 0.808, HD#b: 0.733 Tyr-46 7.59 4.27 HB#a: 2.938, HD#: 7.049, HE#: 6.784 Gln-47 7.49 3.87 HB#a: 2.235, HB#b: 1.948, HG#a: 2.305, HG#b: 2.076 Leu-48 7.66 4.11 HB#a: 1.930, HB#b: 1.409, HG: 1.683; HD#a: 0.677, HD#b: N/A Glu-49 7.75 4.12 HB#a: N/A, HB#b: 1.935, HG#a: 2.291, HG#b: 2.191 Asn-50 7.21 4.34 HB#a: 2.363, HB#b: N/A Tyr-51 7.76 4.50 HB#a: 3.269, HB#b: 2.774, HD#: 7.250, HE#: 6.670 Cys-52 7.35 5.01 HB#a: 2.785, HB#b: 3.322 Asn-53 8.18 4.40 HB#a: 2.555, HB#b: 2.716, HD2#a: 7.426, HD2#b: N/A

TABLE 5 Atomic coordinates of Asp²⁸IP(MetTrpLys) in PDB format ATOM 1 CA PHE 1 −6.075 −6.762 −0.761 1.00 0.00 ATOM 2 HA PHE 1 −5.526 −6.571 0.150 1.00 0.00 ATOM 3 CB PHE 1 −5.359 −6.093 −1.942 1.00 0.00 ATOM 4 HB1 PHE 1 −5.934 −6.258 −2.842 1.00 0.00 ATOM 5 HB2 PHE 1 −4.382 −6.535 −2.061 1.00 0.00 ATOM 6 CG PHE 1 −5.208 −4.597 −1.715 1.00 0.00 ATOM 7 CD1 PHE 1 −4.925 −4.081 −0.436 1.00 0.00 ATOM 8 HD1 PHE 1 −4.817 −4.749 0.405 1.00 0.00 ATOM 9 CD2 PHE 1 −5.346 −3.722 −2.799 1.00 0.00 ATOM 10 HD2 PHE 1 −5.563 −4.112 −3.783 1.00 0.00 ATOM 11 CE1 PHE 1 −4.787 −2.702 −0.251 1.00 0.00 ATOM 12 HE1 PHE 1 −4.573 −2.308 0.732 1.00 0.00 ATOM 13 CE2 PHE 1 −5.204 −2.341 −2.612 1.00 0.00 ATOM 14 HE2 PHE 1 −5.310 −1.668 −3.451 1.00 0.00 ATOM 15 CZ PHE 1 −4.926 −1.831 −1.338 1.00 0.00 ATOM 16 HZ PHE 1 −4.818 −0.768 −1.195 1.00 0.00 ATOM 17 C PHE 1 −7.491 −6.201 −0.628 1.00 0.00 ATOM 18 O PHE 1. −8.361 −6.497 −1.425 1.00 0.00 ATOM 19 N PHE 1 −6.144 −8.234 −0.995 1.00 0.00 ATOM 20 HT1 PHE 1 −6.303 −8.723 −0.091 1.00 0.00 ATOM 21 HT2 PHE 1 −5.250 −8.560 −1.415 1.00 0.00 ATOM 22 HT3 PHE 1 −6.930 −8.446 −1.642 1.00 0.00 ATOM 23 N VAL 2 −7.730 −5.399 0.380 1.00 0.00 ATOM 24 HN VAL 2 −7.013 −5.181 1.011 1.00 0.00 ATOM 25 CA VAL 2 −9.095 −4.819 0.578 1.00 0.00 ATOM 26 HA VAL 2 −9.838 −5.571 0.354 1.00 0.00 ATOM 27 CB VAL 2 −9.270 −4.345 2.030 1.00 0.00 ATOM 28 HB VAL 2 −8.787 −3.387 2.154 1.00 0.00 ATOM 29 CG1 VAL 2 −10.760 −4.206 2.339 1.00 0.00 ATOM 30 HG11 VAL 2 −11.281 −3.863 1.457 1.00 0.00 ATOM 31 HG12 VAL 2 −10.897 −3.493 3.139 1.00 0.00 ATOM 32 HG13 VAL 2 −11.156 −5.164 2.641 1.00 0.00 ATOM 33 CG2 VAL 2 −8.653 −5.362 3.001 1.00 0.00 ATOM 34 HG21 VAL 2 −8.836 −6.363 2.637 1.00 0.00 ATOM 35 HG22 VAL 2 −9.103 −5.245 3.976 1.00 0.00 ATOM 36 HG23 VAL 2 −7.589 −5.193 3.071 1.00 0.00 ATOM 37 C VAL 2 −9.279 −3.628 −0.369 1.00 0.00 ATOM 38 O VAL 2 −8.349 −2.892 −0.637 1.00 0.00 ATOM 39 N ASN 3 −10.473 −3.437 −0.874 1.00 0.00 ATOM 40 HN ASN 3 −11.204 −4.045 −0.641 1.00 0.00 ATOM 41 CA ASN 3 −10.725 −2.296 −1.806 1.00 0.00 ATOM 42 HA ASN 3 −9.782 −1.927 −2.184 1.00 0.00 ATOM 43 CB ASN 3 −11.594 −2.772 −2.975 1.00 0.00 ATOM 44 HB1 ASN 3 −12.168 −1.940 −3.357 1.00 0.00 ATOM 45 HB2 ASN 3 −12.265 −3.545 −2.633 1.00 0.00 ATOM 46 CG ASN 3 −10.702 −3.324 −4.087 1.00 0.00 ATOM 47 OD1 ASN 3 −9.628 −3.832 −3.824 1.00 0.00 ATOM 48 ND2 ASN 3 −11.103 −3.251 −5.327 1.00 0.00 ATOM 49 HD21 ASN 3 −11.968 −2.842 −5.538 1.00 0.00 ATOM 50 HD22 ASN 3 −10.539 −3.603 −6.046 1.00 0.00 ATOM 51 C ASN 3 −11.448 −1.171 −1.055 1.00 0.00 ATOM 52 O ASN 3 −12.648 −1.002 −1.182 1.00 0.00 ATOM 53 N GLN 4 −10.727 −0.404 −0.276 1.00 0.00 ATOM 54 RN GLN 4 −9.763 −0.562 −0.193 1.00 0.00 ATOM 55 CA GLN 4 −11.370 0.711 0.484 1.00 0.00 ATOM 56 HA GLN 4 −12.282 1.008 −0.011 1.00 0.00 ATOM 57 CS GLN 4 −11.690 0.260 1.917 1.00 0.00 ATOM 58 HB1 GLN 4 −12.381 0.958 2.365 1.00 0.00 ATOM 59 HB2 GLN 4 −10.779 0.237 2.498 1.00 0.00 ATOM 60 CG CLN 4 −12.318 −1.136 1.905 1.00 0.00 ATOM 61 HG1 GLN 4 −11.533 −1.879 1.890 1.00 0.00 ATOM 62 HG2 GLN 4 −12.936 −1.245 1.028 1.00 0.00 ATOM 63 CD GLN 4 −13.173 −1.325 3.160 1.00 0.00 ATOM 64 OE1 GLN 4 −13.712 −0.374 3.692 1.00 0.00 ATOM 65 NE2 GLN 4 −13.323 −2.522 3.660 1.00 0.00 ATOM 66 HE21 GLN 4 −12.892 −3.289 3.231 1.00 0.00 ATOM 67 HE22 GLN 4 −13.867 −2.653 4.465 1.00 0.00 ATOM 68 C CLN 4 −10.410 1.903 0.543 1.00 0.00 ATOM 69 O GLN 4 −9.424 1.950 −0.168 1.00 0.00 ATOM 70 N HIS 5 −10.690 2.857 1.392 1.00 0.00 ATOM 71 HN HIS 5 −11.488 2.787 1.958 1.00 0.00 ATOM 72 CA HIS 5 −9.799 4.044 1.516 1.00 0.00 ATOM 73 HA HIS 5 −9.334 4.246 0.562 1.00 0.00 ATOM 74 CB HIS 5 −10.622 5.258 1.951 1.00 0.00 ATOM 75 HB1 HIS 5 −9.995 6.137 1.964 1.00 0.00 ATOM 76 HB2 HIS 5 −11.024 5.087 2.939 1.00 0.00 ATOM 77 CG HIS 5 −11.749 5.459 0.974 1.00 0.00 ATOM 78 ND1 HIS 5 −12.883 4.665 0.982 1.00 0.00 ATOM 79 HD1 HIS 5 −13.076 3.934 1.608 1.00 0.00 ATOM 80 CD2 HIS 5 −11.918 6.343 −0.064 1.00 0.00 ATOM 81 HD2 HIS 5 −11.211 7.103 −0.355 1.00 0.00 ATOM 82 CE1 HIS 5 −13.677 5.080 −0.021 1.00 0.00 ATOM 83 HE1 HIS 5 −14.629 4.635 −0.264 1.00 0.00 ATOM 84 NE2 HIS 5 −13.136 6.102 −0.690 1.00 0.00 ATOM 85 C HIS 5 −8.718 3.742 2.554 1.00 0.00 ATOM 86 O HIS 5 −9.006 3.446 3.697 1.00 0.00 ATOM 87 N LEU 6 −7.475 3.794 2.152 1.00 0.00 ATOM 88 HN LEU 6 −7.277 4.020 1.219 1.00 0.00 ATOM 89 CA LEU 6 −6.360 3.485 3.093 1.00 0.00 ATOM 90 HA LEU 6 −6.687 2.754 3.815 1.00 0.00 ATOM 91 CB LEU 6 −5.180 2.923 2.298 1.00 0.00 ATOM 92 HB1 LEU 6 −4.416 2.580 2.979 1.00 0.00 ATOM 93 HB2 LEU 6 −4.776 3.705 1.667 1.00 0.00 ATOM 94 CG LEU 6 −5.654 1.752 1.428 1.00 0.00 ATOM 95 HG LEU 6 −6.504 2.065 0.841 1.00 0.00 ATOM 96 CD1 LEU 6 −4.523 1.318 0.486 1.00 0.00 ATOM 97 HD11 LEU 6 −3.801 2.116 0.395 1.00 0.00 ATOM 98 HD12 LEU 6 −4.935 1.094 −0.487 1.00 0.00 ATOM 99 HD13 LEU 6 −4.039 0.437 0.881 1.00 0.00 ATOM 100 CD2 LEU 6 −6.058 0.574 2.328 1.00 0.00 ATOM 101 HD21 LEU 6 −5.710 −0.352 1.892 1.00 0.00 ATOM 102 HD22 LEU 6 −7.136 0.544 2.417 1.00 0.00 ATOM 103 HD23 LEU 6 −5.621 0.699 3.308 1.00 0.00 ATOM 104 C LEU 6 −5.912 4.757 3.814 1.00 0.00 ATOM 105 O LEU 6 −5.356 5.654 3.213 1.00 0.00 ATOM 106 N CYS 7 −6.143 4.833 5.101 1.00 0.00 ATOM 107 HN CYS 7 −6.585 4.090 5.563 1.00 0.00 ATOM 108 CA CYS 7 −5.721 6.041 5.870 1.00 0.00 ATOM 109 HA CYS 7 −4.835 6.453 5.422 1.00 0.00 ATOM 110 HB1 CYS 7 −7.133 7.322 6.855 1.00 0.00 ATOM 111 HB2 CYS 7 −7.695 6.688 5.314 1.00 0.00 ATOM 112 C CYS 7 −5.408 5.640 7.314 1.00 0.00 ATOM 113 O CYS 7 −6.280 5.231 8.058 1.00 0.00 ATOM 114 CB CYS 7 −6.844 7.087 5.846 1.00 0.00 ATOM 115 SG CYS 7 −6.272 8.597 5.019 1.00 0.00 ATOM 116 N GLY 8 −4.167 5.760 7.712 1.00 0.00 ATOM 117 HN GLY 8 −3.488 6.094 7.090 1.00 0.00 ATOM 118 CA GLY 8 −3.781 5.394 9.105 1.00 0.00 ATOM 119 HA1 GLY 8 −4.671 5.213 9.690 1.00 0.00 ATOM 120 HA2 GLY 8 −3.219 6.205 9.546 1.00 0.00 ATOM 121 C GLY 8 −2.923 4.129 9.085 1.00 0.00 ATOM 122 O GLY 8 −1.949 4.041 8.360 1.00 0.00 ATOM 123 N SER 9 −3.278 3.150 9.877 1.00 0.00 ATOM 124 HN SER 9 −4.068 3.251 10.451 1.00 0.00 ATOM 125 CA SER 9 −2.490 1.883 9.916 1.00 0.00 ATOM 126 HA SER 9 −1.437 2.116 9.986 1.00 0.00 ATOM 127 CB SER 9 −2.912 1.063 11.135 1.00 0.00 ATOM 128 HB1 SER 9 −3.777 0.464 10.881 1.00 0.00 ATOM 129 HB2 SER 9 −3.164 1.724 11.947 1.00 0.00 ATOM 130 OG SER 9 −1.837 0.222 11.531 1.00 0.00 ATOM 131 HG SER 9 −1.789 −0.510 10.913 1.00 0.00 ATOM 132 C SER 9 −2.751 1.076 8.643 1.00 0.00 ATOM 133 O SER 9 −1.897 0.350 8.173 1.00 0.00 ATOM 134 N HIS 10 −3.929 1.197 8.088 1.00 0.00 ATOM 135 HN HIS 10 −4.599 1.790 8.489 1.00 0.00 ATOM 136 CA HIS 10 −4.258 0.439 6.843 1.00 0.00 ATOM 137 HA HIS 10 −4.122 −0.614 7.021 1.00 0.00 ATOM 138 CB HIS 10 −5.720 0.706 6.456 1.00 0.00 ATOM 139 HB1 HIS 10 −5.771 0.967 5.413 1.00 0.00 ATOM 140 HB2 HIS 10 −6.107 1.522 7.049 1.00 0.00 ATOM 141 CG HIS 10 −6.547 −0.528 6.700 1.00 0.00 ATOM 142 ND1 HIS 10 −6.371 −1.687 5.963 1.00 0.00 ATOM 143 HD1 HIS 10 −5.723 −1.822 5.241 1.00 0.00 ATOM 144 CD2 HIS 10 −7.556 −0.797 7.591 1.00 0.00 ATOM 145 HD2 HIS 10 −7.945 −0.101 8.319 1.00 0.00 ATOM 146 CE1 HIS 10 −7.253 −2.594 6.419 1.00 0.00 ATOM 147 HE1 HIS 10 −7.345 −3.595 6.027 1.00 0.00 ATOM 148 NE2 HIS 10 −8.001 −2.104 7.412 1.00 0.00 ATOM 149 C HIS 10 −3.332 0.880 5.701 1.00 0.00 ATOM 150 O HIS 10 −3.149 0.160 4.737 1.00 0.00 ATOM 151 N LEU 11 −2.755 2.052 5.797 1.00 0.00 ATOM 152 HN LEU 11 −2.922 2.619 6.578 1.00 0.00 ATOM 153 CA LEU 11 −1.851 2.536 4.713 1.00 0.00 ATOM 154 HA LEU 11 −2.384 2.534 3.774 1.00 0.00 ATOM 155 CB LEU 11 −1.392 3.961 5.031 1.00 0.00 ATOM 156 HB1 LEU 11 −0.726 3.942 5.880 1.00 0.00 ATOM 157 HB2 LEU 11 −2.253 4.572 5.261 1.00 0.00 ATOM 158 CG LEU 11 −0.658 4.543 3.823 1.00 0.00 ATOM 159 HG LEU 11 0.144 3.879 3.533 1.00 0.00 ATOM 160 CD1 LEU 11 −1.635 4.701 2.655 1.00 0.00 ATOM 161 HD11 LEU 11 −2.565 5.114 3.017 1.00 0.00 ATOM 162 HD12 LEU 11 −1.821 3.735 2.209 1.00 0.00 ATOM 163 HD13 LEU 11 −1.209 5.362 1.916 1.09 0.00 ATOM 164 CD2 LEU 11 −0.082 5.912 4.194 1.00 0.00 ATOM 165 HD21 LEU 11 −0.855 6.660 4.114 1.00 0.00 ATOM 166 HD22 LEU 11 0.727 6.156 3.522 1.00 0.00 ATOM 167 HD23 LEU 11 0.288 5.883 5.207 1.00 0.00 ATOM 168 C LEU 11 −0.628 1.620 4.604 1.00 0.00 ATOM 169 O LEU 11 −0.303 1.133 3.539 1.00 0.00 ATOM 170 N VAL 12 0.055 1.390 5.699 1.00 0.00 ATOM 171 HN VAL 12 −0.226 1.800 6.543 1.00 0.00 ATOM 172 CA VAL 12 1.265 0.514 5.663 1.00 0.00 ATOM 173 HA VAL 12 1.903 0.819 4.847 1.00 0.00 ATOM 174 GB VAL 12 2.033 0.651 6.981 1.00 0.00 ATOM 175 HB VAL 12 1.442 0.243 7.787 1.00 0.00 ATOM 176 CG1 VAL 12 3.356 −0.110 6.883 1.00 0.00 ATOM 177 HG11 VAL 12 3.936 0.059 7.777 1.00 0.00 ATOM 178 HG12 VAL 12 3.909 0.239 6.023 1.00 0.00 ATOM 179 HG13 VAL 12 3.157 −1.167 6.778 1.00 0.00 ATOM 180 CG2 VAL 12 2.321 2.130 7.253 1.00 0.00 ATOM 181 HG21 VAL 12 2.917 2.534 6.446 1.00 0.00 ATOM 182 HG22 VAL 12 2.862 2.225 8.182 1.00 0.00 ATOM 183 HG23 VAL 12 1.390 2.673 7.322 1.00 0.00 ATOM 184 C VAL 12 0.852 −0.950 5.461 1.00 0.00 ATOM 185 O VAL 12 1.637 −1.764 5.009 1.00 0.00 ATOM 186 N GLU 13 −0.365 −1.294 5.802 1.00 0.00 ATOM 187 HN GLU 13 −0.978 −0.625 6.171 1.00 0.00 ATOM 188 CA GLU 13 −0.827 −2.706 5.640 1.00 0.00 ATOM 189 HA GLU 13 −0.099 −3.371 6.080 1.00 0.00 ATOM 190 CB GLU 13 −2.165 −2.882 6.361 1.00 0.00 ATOM 191 HB1 GLU 13 −2.707 −3.703 5.916 1.00 0.00 ATOM 192 HB2 GLU 13 −2.745 −1.975 6.270 1.00 0.00 ATOM 193 CG GLU 13 −1.913 −3.185 7.840 1.00 0.00 ATOM 194 HG1 GLU 13 −2.783 −2.910 8.417 1.00 0.00 ATOM 195 HG2 GLU 13 −1.060 −2.619 8.182 1.00 0.00 ATOM 196 CD GLU 13 −1.638 −4.680 8.016 1.00 0.00 ATOM 197 OE1 GLU 13 −2.032 −5.217 9.038 1.00 0.00 ATOM 198 OE2 GLU 13 −1.036 −5.261 7.128 1.00 0.00 ATOM 199 C GLU 13 −1.001 −3.058 4.154 1.00 0.00 ATOM 200 O GLU 13 −1.130 −4.219 3.805 1.00 0.00 ATOM 201 N ALA 14 −1.020 −2.079 3.277 1.00 0.00 ATOM 202 HN ALA 14 −0.923 −1.151 3.574 1.00 0.00 ATOM 203 CA ALA 14 −1.200 −2.381 1.824 1.00 0.00 ATOM 204 HA ALA 14 −1.699 −3.333 1.714 1.00 0.00 ATOM 205 CB ALA 14 −2.055 −1.288 1.178 1.00 0.00 ATOM 206 HB1 ALA 14 −2.984 −1.191 1.722 1.00 0.00 ATOM 207 HB2 ALA 14 −2.266 −1.556 0.152 1.00 0.00 ATOM 208 HB3 ALA 14 −1.522 −0.350 1.203 1.00 0.00 ATOM 209 C ALA 14 0.160 −2.443 1.120 1.00 0.00 ATOM 210 O ALA 14 0.340 −3.189 0.176 1.00 0.00 ATOM 211 N LEU 15 1.114 −1.662 1.565 1.00 0.00 ATOM 212 HN LEU 15 0.943 −1.065 2.324 1.00 0.00 ATOM 213 CA LEU 15 2.459 −1.675 0.914 1.00 0.00 ATOM 214 HA LEU 15 2.338 −1.551 −0.153 1.00 0.00 ATOM 215 GB LEU 15 3.309 −0.529 1.462 1.00 0.00 ATOM 216 HB1 LEU 15 4.336 −0.661 1.152 1.00 0.00 ATOM 217 HB2 LEU 15 3.256 −0.525 2.542 1.00 0.00 ATOM 218 CG LEU 15 2.785 0.801 0.921 1.00 0.00 ATOM 219 HG LEU 15 1.729 0.885 1.137 1.00 0.00 ATOM 220 CD1 LEU 15 3.533 1.956 1.590 1.00 0.00 ATOM 221 HD11 LEU 15 3.903 1.635 2.554 1.00 0.00 ATOM 222 HD12 LEU 15 2.863 2.791 1.723 1.00 0.00 ATOM 223 HD13 LEU 15 4.364 2.256 0.969 1.00 0.00 ATOM 224 CD2 LEU 15 3.006 0.859 −0.594 1.00 0.00 ATOM 225 HD21 LEU 15 2.950 1.884 −0.927 1.00 0.00 ATOM 226 HD22 LEU 15 2.243 0.278 −1.092 1.00 0.00 ATOM 227 HD23 LEU 15 3.978 0.456 −0.834 1.00 0.00 ATOM 228 C LEU 15 3.156 −3.009 1.190 1.00 0.00 ATOM 229 O LEU 15 3.865 −3.528 0.349 1.00 0.00 ATOM 230 N TYR 16 2.957 −3.571 2.358 1.00 0.00 ATOM 231 HN TYR 16 2.378 −3.135 3.018 1.00 0.00 ATOM 232 CA TYR 16 3.606 −4.876 2.681 1.00 0.00 ATOM 233 HA TYR 16 4.609 −4.869 2.277 1.00 0.00 ATOM 234 CB TYR 16 3.689 −5.037 4.230 1.00 0.00 ATOM 235 HB1 TYR 16 3.494 −4.076 4.686 1.00 0.00 ATOM 236 HB2 TYR 16 4.688 −5.346 4.492 1.00 0.00 ATOM 237 CG TYR 16 2.704 −6.057 4.783 1.00 0.00 ATOM 238 CD1 TYR 16 3.181 −7.209 5.421 1.00 0.00 ATOM 239 HD1 TYR 16 4.244 −7.371 5.517 1.00 0.00 ATOM 240 CD2 TYR 16 1.324 −5.846 4.659 1.00 0.00 ATOM 241 HD2 TYR 16 0.954 −4.960 4.166 1.00 0.00 ATOM 242 CE1 TYR 16 2.282 −8.150 5.934 1.00 0.00 ATOM 243 HE1 TYR 16 2.651 −9.038 6.425 1.00 0.00 ATOM 244 CE2 TYR 16 0.423 −6.790 5.171 1.00 0.00 ATOM 245 HE2 TYR 16 −0.640 −6.628 5.076 1.00 0.00 ATOM 246 CZ TYR 16 0.902 −7.941 5.809 1.00 0.00 ATOM 247 OH TYR 16 0.016 −8.868 6.315 1.00 0.00 ATOM 248 HH TYR 16 −0.479 −8.451 7.024 1.00 0.00 ATOM 249 C TYR 16 2.815 −6.006 1.997 1.00 0.00 ATOM 250 O TYR 16 3.363 −7.026 1.626 1.00 0.00 ATOM 251 N LEU 17 1.531 −5.816 1.825 1.00 0.00 ATOM 252 HN LEU 17 1.118 −4.981 2.129 1.00 0.00 ATOM 253 CA LEU 17 0.691 −6.853 1.160 1.00 0.00 ATOM 254 HA LEU 17 0.816 −7.799 1.665 1.00 0.00 ATOM 255 CB LEU 17 −0.778 −6.421 1.227 1.00 0.00 ATOM 256 HB1 LEU 17 −0.897 −5.483 0.705 1.00 0.00 ATOM 257 HB2 LEU 17 −1.068 −6.295 2.260 1.00 0.00 ATOM 258 CG LEU 17 −1.668 −7.480 0.573 1.00 0.00 ATOM 259 HG LEU 17 −1.242 −7.773 −0.377 1.00 0.00 ATOM 260 CD1 LEU 17 −1.767 −8.704 1.484 1.00 0.00 ATOM 261 HD11 LEU 17 −2.581 −9.332 1.154 1.00 0.00 ATOM 262 HD12 LEU 17 −1.950 −8.384 2.499 1.00 0.00 ATOM 263 HD13 LEU 17 −0.844 −9.260 1.442 1.00 0.00 ATOM 264 CD2 LEU 17 −3.064 −6.898 0.350 1.00 0.00 ATOM 265 HD21 LEU 17 −3.522 −7.382 −0.499 1.00 0.00 ATOM 266 HD22 LEU 17 −2.985 −5.838 0.163 1.00 0.00 ATOM 267 HD23 LEU 17 −3.668 −7.065 1.228 1.00 0.00 ATOM 268 C LEU 17 1.128 −6.987 −0.302 1.00 0.00 ATOM 269 O LEU 17 1.024 −8.044 −0.897 1.00 0.00 ATOM 270 N VAL 18 1.614 −5.919 −0.883 1.00 0.00 ATOM 271 HN VAL 18 1.683 −5.081 −0.380 1.00 0.00 ATOM 272 CA VAL 18 2.059 −5.966 −2.306 1.00 0.00 ATOM 273 HA VAL 18 1.392 −6.595 −2.875 1.00 0.00 ATOM 274 CB VAL 18 2.052 −4.548 −2.886 1.00 0.00 ATOM 275 HB VAL 18 2.802 −3.954 −2.382 1.00 0.00 ATOM 276 CG1 VAL 18 2.375 −4.606 −4.380 1.00 0.00 ATOM 277 HG11 VAL 18 1.457 −4.614 −4.947 1.00 0.00 ATOM 278 HG12 VAL 18 2.939 −5.504 −4.594 1.00 0.00 ATOM 279 HG13 VAL 18 2.961 −3.741 −4.656 1.00 0.00 ATOM 280 CG2 VAL 18 0.674 −3.907 −2.683 1.00 0.00 ATOM 281 HG21 VAL 18 0.117 −4.466 −1.943 1.00 0.00 ATOM 282 HG22 VAL 18 0.131 −3.910 −3.617 1.00 0.00 ATOM 283 HG23 VAL 18 0.799 −2.889 −2.345 1.00 0.00 ATOM 284 C VAL 18 3.480 −6.523 −2.376 1.00 0.00 ATOM 285 O VAL 18 3.801 −7.325 −3.233 1.00 0.00 ATOM 286 N CYS 19 4.334 −6.086 −1.490 1.00 0.00 ATOM 287 HN CYS 19 4.048 −5.430 −0.821 1.00 0.00 ATOM 288 CA CYS 19 5.746 −6.566 −1.501 1.00 0.00 ATOM 289 HA CYS 19 5.981 −6.963 −2.478 1.00 0.00 ATOM 290 HB1 CYS 19 7.708 −5.690 −1.396 1.00 0.00 ATOM 291 HB2 CYS 19 6.603 −5.126 −0.146 1.00 0.00 ATOM 292 C CYS 19 5.910 −7.669 −0.451 1.00 0.00 ATOM 293 O CYS 19 6.116 −8.822 −0.782 1.00 0.00 ATOM 294 CB CYS 19 6.693 −5.394 −1.186 1.00 0.00 ATOM 295 SG CYS 19 6.271 −3.960 −2.210 1.00 0.00 ATOM 296 N GLY 20 5.806 −7.326 0.809 1.00 0.00 ATOM 297 HN GLY 20 5.629 −6.392 1.048 1.00 0.00 ATOM 298 CA GLY 20 5.938 −8.354 1.885 1.00 0.00 ATOM 299 HA1 GLY 20 5.615 −9.313 1.508 1.00 0.00 ATOM 300 HA2 GLY 20 5.319 −8.074 2.725 1.00 0.00 ATOM 301 C GLY 20 7.395 −8.458 2.341 1.00 0.00 ATOM 302 O GLY 20 7.969 −7.508 2.837 1.00 0.00 ATOM 303 N GLU 21 7.991 −9.615 2.186 1.00 0.00 ATOM 304 HN GLU 21 7.499 −10.364 1.789 1.00 0.00 ATOM 305 CA GLU 21 9.410 −9.809 2.615 1.00 0.00 ATOM 306 HA GLU 21 9.482 −9.667 3.684 1.00 0.00 ATOM 307 CB GLU 21 9.856 −11.229 2.262 1.00 0.00 ATOM 308 HB1 GLU 21 10.925 −11.314 2.391 1.00 0.00 ATOM 309 HB2 GLU 21 9.598 −11.441 1.235 1.00 0.00 ATOM 310 CG GLU 21 9.154 −12.227 3.183 1.00 0.00 ATOM 311 HG1 GLU 21 8.975 −13.149 2.647 1.00 0.00 ATOM 312 HG2 GLU 21 8.212 −11.813 3.513 1.00 0.00 ATOM 313 CD GLU 21 10.040 −12.513 4.398 1.00 0.00 ATOM 314 OE1 GLU 21 9.858 −11.851 5.407 1.00 0.00 ATOM 315 OE2 GLU 21 10.885 −13.387 4.298 1.00 0.00 ATOM 316 C GLU 21 10.321 −8.800 1.908 1.00 0.00 ATOM 317 O GLU 21 11.338 −8.394 2.441 1.00 0.00 ATOM 318 N ARG 22 9.965 −8.395 0.715 1.00 0.00 ATOM 319 HN ARG 22 9.141 −8.739 0.310 1.00 0.00 ATOM 320 CA ARG 22 10.807 −7.413 −0.030 1.00 0.00 ATOM 321 HA ARG 22 11.805 −7.809 −0.145 1.00 0.00 ATOM 322 CB ARG 22 10.198 −7.162 −1.410 1.00 0.00 ATOM 323 HB1 ARG 22 10.595 −6.244 −1.813 1.00 0.00 ATOM 324 HB2 ARG 22 9.126 −7.079 −1.314 1.00 0.00 ATOM 325 CG ARG 22 10.537 −8.329 −2.351 1.00 0.00 ATOM 326 HG1 ARG 22 9.626 −8.727 −2.767 1.00 0.00 ATOM 327 HG2 ARG 22 11.050 −9.104 −1.801 1.00 0.00 ATOM 328 CD ARG 22 11.438 −7.835 −3.489 1.00 0.00 ATOM 329 HD1 ARG 22 10.951 −7.021 −4.006 1.00 0.00 ATOM 330 HD2 ARG 22 11.618 −8.644 −4.182 1.00 0.00 ATOM 331 NE ARG 22 12.735 −7.363 −2.928 1.00 0.00 ATOM 332 HE ARG 22 12.747 −6.827 −2.107 1.00 0.00 ATOM 333 CZ ARG 22 13.854 −7.662 −3.527 1.00 0.00 ATOM 334 NH1 ARG 22 13.963 −7.517 −4.820 1.00 0.00 ATOM 335 HH11 ARG 22 13.186 −7.175 −5.351 1.00 0.00 ATOM 336 HH12 ARG 22 14.821 −7.746 −5.280 1.00 0.00 ATOM 337 NH2 ARG 22 14.867 −8.105 −2.835 1.00 0.00 ATOM 338 HH21 ARG 22 14.785 −8.216 −1.845 1.00 0.00 ATOM 339 HH22 ARG 22 15.726 −8.335 −3.294 1.00 0.00 ATOM 340 C ARG 22 10.870 −6.098 0.750 1.00 0.00 ATOM 341 O ARG 22 11.919 −5.497 0.889 1.00 0.00 ATOM 342 N GLY 23 9.752 −5.652 1.266 1.00 0.00 ATOM 343 HN GLY 23 8.923 −6.159 1.141 1.00 0.00 ATOM 344 CA GLY 23 9.734 −4.378 2.045 1.00 0.00 ATOM 345 HA1 GLY 23 10.693 −4.233 2.5201 1.00 0.00 ATOM 346 HA2 GLY 23 8.962 −4.434 2.800 1.00 0.00 ATOM 347 C GLY 23 9.450 −3.200 1.111 1.00 0.00 ATOM 348 O GLY 23 9.449 −3.339 −0.098 1.00 0.00 ATOM 349 N PHE 24 9.207 −2.041 1.668 1.00 0.00 ATOM 350 HN PHE 24 9.214 −1.961 2.646 1.00 0.00 ATOM 351 CA PHE 24 8.919 −0.838 0.829 1.00 0.00 ATOM 352 HA PHE 24 9.341 −0.984 −0.155 1.00 0.00 ATOM 353 CB PHE 24 7.400 −0.629 0.703 1.00 0.00 ATOM 354 HB1 PHE 24 7.001 −1.341 −0.004 1.00 0.00 ATOM 355 HB2 PHE 24 7.203 0.372 0.350 1.00 0.00 ATOM 356 CG PHE 24 6.723 −0.828 2.043 1.00 0.00 ATOM 357 CD1 PHE 24 6.408 −2.122 2.472 1.00 0.00 ATOM 358 HD1 PHE 24 6.652 −2.968 1.848 1.00 0.00 ATOM 359 CD2 PHE 24 6.406 0.272 2.848 1.00 0.00 ATOM 360 HD2 PHE 24 6.648 1.272 2.520 1.00 0.00 ATOM 361 CE1 PHE 24 5.778 −2.318 3.706 1.00 0.00 ATOM 362 HE1 PHE 24 5.537 −3.318 4.036 1.00 0.00 ATOM 363 CE2 PHE 24 5.776 0.077 4.083 1.00 0.00 ATOM 364 HE2 PHE 24 5.531 0.926 4.706 1.00 0.00 ATOM 365 CZ PHE 24 5.461 −1.220 4.513 1.00 0.00 ATOM 366 HZ PHE 24 4.975 −1.370 5.464 1.00 0.00 ATOM 367 C PHE 24 9.557 0.396 1.472 1.00 0.00 ATOM 368 O PHE 24 9.004 0.992 2.376 1.00 0.00 ATOM 369 N PHE 25 10.722 0.780 1.010 1.00 0.00 ATOM 370 HN PHE 25 11.146 0.278 0.283 1.00 0.00 ATOM 371 CA PHE 25 11.408 1.972 1.590 1.00 0.00 ATOM 372 HA PHE 25 11.138 2.074 2.631 1.00 0.00 ATOM 373 CB PHE 25 12.924 1.791 1.474 1.00 0.00 ATOM 374 HB1 PHE 25 13.411 2.748 1.591 1.00 0.00 ATOM 375 HB2 PHE 25 13.163 1.382 0.503 1.00 0.00 ATOM 376 CG PHE 25 13.407 0.848 2.551 1.00 0.00 ATOM 377 CD1 PHE 25 13.976 −0.381 2.200 1.00 0.00 ATOM 378 HD1 PHE 25 14.069 −0.657 1.161 1.00 0.00 ATOM 379 CD2 PHE 25 13.284 1.206 3.898 1.00 0.00 ATOM 380 HD2 PHE 25 12.845 2.154 4.169 1.00 0.00 ATOM 381 CE1 PHE 25 14.423 −1.255 3.197 1.00 0.00 ATOM 382 HE1 PHE 25 14.864 −2.204 2.927 1.00 0.00 ATOM 383 CE2 PHE 25 13.732 0.332 4.896 1.00 0.00 ATOM 384 HE2 PHE 25 13.639 0.606 5.937 1.00 0.00 ATOM 385 CZ PHE 25 14.302 −0.900 4.545 1.00 0.00 ATOM 386 HZ PHE 25 14.648 −1.573 5.315 1.00 0.00 ATOM 387 C PHE 25 10.991 3.230 0.826 1.00 0.00 ATOM 388 O PHE 25 10.374 3.155 −0.220 1.00 0.00 ATOM 389 N TYR 26 11.325 4.386 1.345 1.00 0.00 ATOM 390 HN TYR 26 11.823 4.414 2.188 1.00 0.00 ATOM 391 CA TYR 26 10.956 5.659 0.659 1.00 0.00 ATOM 392 HA TYR 26 10.108 5.486 0.011 1.00 0.00 ATOM 393 CB TYR 26 10.594 6.719 1.703 1.00 0.00 ATOM 394 HB1 TYR 26 10.213 7.598 1.205 1.00 0.00 ATOM 395 HB2 TYR 26 11.475 6.980 2.270 1.00 0.00 ATOM 396 CG TYR 26 9.539 6.173 2.635 1.00 0.00 ATOM 397 CD1 TYR 26 8.289 5.794 2.131 1.00 0.00 ATOM 398 HD1 TYR 26 8.079 5.891 1.076 1.00 0.00 ATOM 399 CD2 TYR 26 9.809 6.046 4.002 1.00 0.00 ATOM 400 HD2 TYR 26 10.772 6.339 4.392 1.00 0.00 ATOM 401 CE1 TYR 26 7.311 5.287 2.993 1.00 0.00 ATOM 402 HE1 TYR 26 6.348 4.994 2.604 1.00 0.00 ATOM 403 CE2 TYR 26 8.829 5.540 4.866 1.00 0.00 ATOM 404 HE2 TYR 26 9.038 5.442 5.922 1.00 0.00 ATOM 405 CZ TYR 26 7.581 5.161 4.362 1.00 0.00 ATOM 406 OH TYR 26 6.616 4.661 5.212 1.00 0.00 ATOM 407 HH TYR 26 6.614 3.707 5.129 1.00 0.00 ATOM 408 C TYR 26 12.143 6.150 −0.171 1.00 0.00 ATOM 409 O TYR 26 13.211 5.565 −0.147 1.00 0.00 ATOM 410 N THR 27 11.966 7.221 −0.907 1.00 0.00 ATOM 411 HN THR 27 11.096 7.673 −0.908 1.00 0.00 ATOM 412 CA THR 27 13.084 7.753 −1.741 1.00 0.00 ATOM 413 HA THR 27 14.023 7.574 −1.237 1.00 0.00 ATOM 414 CB THR 27 13.094 7.039 −3.097 1.00 0.00 ATOM 415 HB THR 27 13.715 7.589 −3.787 1.00 0.00 ATOM 416 OG1 THR 27 11.770 6.969 −3.604 1.00 0.00 ATOM 417 HG1 THR 27 11.816 6.651 −4.509 1.00 0.00 ATOM 418 CG2 THR 27 13.657 5.627 −2.928 1.00 0.00 ATOM 419 HG21 THR 27 13.880 5.211 −3.899 1.00 0.00 ATOM 420 HG22 THR 27 12.925 5.007 −2.429 1.00 0.00 ATOM 421 HG23 THR 27 14.559 5.666 −2.337 1.00 0.00 ATOM 422 C THR 27 12.906 9.258 −1.960 1.00 0.00 ATOM 423 O THR 27 13.757 10.048 −1.596 1.00 0.00 ATOM 424 N ASP 28 11.813 9.660 −2.564 1.00 0.00 ATOM 425 HN ASP 28 11.147 9.003 −2.857 1.00 0.00 ATOM 426 CA ASP 28 11.588 11.115 −2.820 1.00 0.00 ATOM 427 HA ASP 28 12.312 11.692 −2.264 1.00 0.00 ATOM 428 CB ASP 28 11.759 11.396 −4.315 1.00 0.00 ATOM 429 HB1 ASP 28 11.472 12.415 −4.524 1.00 0.00 ATOM 430 HB2 ASP 28 11.133 10.722 −4.881 1.00 0.00 ATOM 431 CG ASP 28 13.222 11.188 −4.712 1.00 0.00 ATOM 432 OD1 ASP 28 13.942 12.170 −4.780 1.00 0.00 ATOM 433 OD2 ASP 28 13.597 10.050 −4.941 1.00 0.00 ATOM 434 C ASP 28 10.176 11.519 −2.384 1.00 0.00 ATOM 435 O ASP 28 9.993 12.153 −1.363 1.00 0.00 ATOM 436 N LYS 29 9.177 11.162 −3.157 1.00 0.00 ATOM 437 HN LYS 29 9.353 10.656 −3.978 1.00 0.00 ATOM 438 CA LYS 29 7.776 11.533 −2.797 1.00 0.00 ATOM 439 HA LYS 29 7.756 11.902 −1.782 1.00 0.00 ATOM 440 CB LYS 29 7.278 12.631 −3.746 1.00 0.00 ATOM 441 HB1 LYS 29 7.869 13.523 −3.607 1.00 0.00 ATOM 442 HB2 LYS 29 6.242 12.850 −3.528 1.00 0.00 ATOM 443 CG LYS 29 7.404 12.159 −5.200 1.00 0.00 ATOM 444 HG1 LYS 29 6.628 11.440 −5.413 1.00 0.00 ATOM 445 HG2 LYS 29 8.370 11.697 −5.344 1.00 0.00 ATOM 446 CD LYS 29 7.258 13.356 −6.151 1.00 0.00 ATOM 447 HD1 LYS 29 6.898 14.215 −5.604 1.00 0.00 ATOM 448 HD2 LYS 29 6.553 13.107 −6.931 1.00 0.00 ATOM 449 CE LYS 29 8.613 13.691 −6.780 1.00 0.00 ATOM 450 HE1 LYS 29 9.391 13.596 −6.036 1.00 0.00 ATOM 451 HE2 LYS 29 8.596 14.702 −7.156 1.00 0.00 ATOM 452 NZ LYS 29 8.885 12.749 −7.905 1.00 0.00 ATOM 453 HZ1 LYS 29 8.366 13.060 −8.750 1.00 0.00 ATOM 454 HZ2 LYS 29 8.574 11.792 −7.638 1.00 0.00 ATOM 455 HZ3 LYS 29 9.904 12.739 −8.110 1.00 0.00 ATOM 456 C LYS 29 6.874 10.299 −2.904 1.00 0.00 ATOM 457 O LYS 29 5.911 10.280 −3.649 1.00 0.00 ATOM 458 N MET 30 7.179 9.270 −2.156 1.00 0.00 ATOM 459 HN MET 30 7.957 9.312 −1.562 1.00 0.00 ATOM 460 CA MET 30 6.346 8.032 −2.201 1.00 0.00 ATOM 461 HA MET 30 5.882 7.948 −3.171 1.00 0.00 ATOM 462 CB MET 30 7.237 6.813 −1.957 1.00 0.00 ATOM 463 HB1 MET 30 7.274 6.597 −0.900 1.00 0.00 ATOM 464 HB2 MET 30 8.235 7.018 −2.319 1.00 0.00 ATOM 465 CG MET 30 6.664 5.607 −2.701 1.00 0.00 ATOM 466 HG1 MET 30 6.455 5.880 −3.724 1.00 0.00 ATOM 467 HG2 MET 30 5.751 5.288 −2.219 1.00 0.00 ATOM 468 SD MET 30 7.863 4.254 −2.670 1.00 0.00 ATOM 469 CE MET 30 7.265 3.464 −1.156 1.00 0.00 ATOM 470 HE1 MET 30 6.460 4.055 −0.737 1.00 0.00 ATOM 471 HE2 MET 30 8.069 3.399 −0.441 1.00 0.00 ATOM 472 HE3 MET 30 6.907 2.471 −1.387 1.00 0.00 ATOM 473 C MET 30 5.260 8.094 −1.119 1.00 0.00 ATOM 474 O MET 30 4.242 7.434 −1.221 1.00 0.00 ATOM 475 N TRP 31 5.469 8.873 −0.086 1.00 0.00 ATOM 476 HN TRP 31 6.298 9.389 −0.024 1.00 0.00 ATOM 477 CA TRP 31 4.455 8.970 1.007 1.00 0.00 ATOM 478 HA TRP 31 3.952 8.022 1.116 1.00 0.00 ATOM 479 CB TRP 31 5.158 9.324 2.319 1.00 0.00 ATOM 480 HB1 TRP 31 5.222 10.397 2.414 1.00 0.00 ATOM 481 HB2 TRP 31 6.154 8.903 2.317 1.00 0.00 ATOM 482 CG TRP 31 4.385 8.766 3.468 1.00 0.00 ATOM 483 CD1 TRP 31 3.902 9.490 4.503 1.00 0.00 ATOM 484 HD1 TRP 31 4.010 10.557 4.630 1.00 0.00 ATOM 485 CD2 TRP 31 3.999 7.384 3.719 1.00 0.00 ATOM 486 NE1 TRP 31 3.246 8.639 5.376 1.00 0.00 ATOM 487 HE1 TRP 31 2.807 8.913 6.208 1.00 0.00 ATOM 488 CE2 TRP 31 3.277 7.331 4.935 1.00 0.00 ATOM 489 CE3 TRP 31 4.203 6.185 3.016 1.00 0.00 ATOM 490 HE3 TRP 31 4.748 6.195 2.084 1.00 0.00 ATOM 491 CZ2 TRP 31 2.778 6.127 5.435 1.00 0.00 ATOM 492 HZ2 TRP 31 2.231 6.112 6.367 1.00 0.00 ATOM 493 CZ3 TRP 31 3.703 4.972 3.516 1.00 0.00 ATOM 494 HZ3 TRP 31 3.866 4.056 2.968 1.00 0.00 ATOM 495 CH2 TRP 31 2.991 4.944 4.724 1.00 0.00 ATOM 496 HH2 TRP 31 2.610 4.007 5.104 1.00 0.00 ATOM 497 C TRP 31 3.421 10.058 0.691 1.00 0.00 ATOM 498 O TRP 31 2.338 10.065 1.243 1.00 0.00 ATOM 499 N LYS 32 3.745 10.983 −0.178 1.00 0.00 ATOM 500 HN LYS 32 4.625 10.969 −0.605 1.00 0.00 ATOM 501 CA LYS 32 2.775 12.072 −0.503 1.00 0.00 ATOM 502 HA LYS 32 2.200 12.313 0.378 1.00 0.00 ATOM 503 CB LYS 32 3.542 13.314 −0.962 1.00 0.00 ATOM 504 HB1 LYS 32 3.751 13.236 −2.019 1.00 0.00 ATOM 505 HB2 LYS 32 4.473 13.381 −0.417 1.00 0.00 ATOM 506 CG LYS 32 2.700 14.570 −0.696 1.00 0.00 ATOM 507 HG1 LYS 32 3.132 15.122 0.125 1.00 0.00 ATOM 508 HG2 LYS 32 1.690 14.284 −0.442 1.00 0.00 ATOM 509 CD LYS 32 2.679 15.456 −1.946 1.00 0.00 ATOM 510 HD1 LYS 32 1.794 16.073 −1.933 1.00 0.00 ATOM 511 HD2 LYS 32 2.669 14.831 −2.826 1.00 0.00 ATOM 512 CE LYS 32 3.923 16.347 −1.966 1.00 0.00 ATOM 513 HE1 LYS 32 4.337 16.417 −0.971 1.00 0.00 ATOM 514 HE2 LYS 32 3.652 17.333 −2.314 1.00 0.00 ATOM 515 NZ LYS 32 4.935 15.758 −2.887 1.00 0.00 ATOM 516 HZ1 LYS 32 5.633 16.483 −3.145 1.00 0.00 ATOM 517 HZ2 LYS 32 4.461 15.410 −3.745 1.00 0.00 ATOM 518 HZ3 LYS 32 5.418 14.970 −2.412 1.00 0.00 ATOM 519 C LYS 32 1.829 11.620 −1.617 1.00 0.00 ATOM 520 O LYS 32 0.658 11.951 −1.616 1.00 0.00 ATOM 521 N GLY 33 2.328 10.879 −2.574 1.00 0.00 ATOM 522 HN GLY 33 3.277 10.635 −2.556 1.00 0.00 ATOM 523 CA GLY 33 1.463 10.419 −3.697 1.00 0.00 ATOM 524 HA1 GLY 33 2.079 10.208 −4.559 1.00 0.00 ATOM 525 HA2 GLY 33 0.756 11.198 −3.947 1.00 0.00 ATOM 526 C GLY 33 0.698 9.149 −3.306 1.00 0.00 ATOM 527 O GLY 33 −0.296 8.811 −3.921 1.00 0.00 ATOM 528 N ILE 34 1.156 8.431 −2.306 1.00 0.00 ATOM 529 HN ILE 34 1.965 8.711 −1.830 1.00 0.00 ATOM 530 CA ILE 34 0.449 7.175 −1.908 1.00 0.00 ATOM 531 HA ILE 34 0.084 6.687 −2.798 1.00 0.00 ATOM 532 CB ILE 34 1.427 6.230 −1.189 1.00 0.00 ATOM 533 HB ILE 34 2.272 6.041 −1.832 1.00 0.00 ATOM 534 CG1 ILE 34 0.714 4.903 −0.882 1.00 0.00 ATOM 535 HG11 ILE 34 0.348 4.473 −1.803 1.00 0.00 ATOM 536 HG12 ILE 34 −0.119 5.089 −0.218 1.00 0.00 ATOM 537 CG2 ILE 34 1.922 6.864 0.119 1.00 0.00 ATOM 538 HG21 ILE 34 1.094 6.969 0.804 1.00 0.00 ATOM 539 HG22 ILE 34 2.346 7.835 −0.087 1.00 0.00 ATOM 540 HG23 ILE 34 2.677 6.228 0.561 1.00 0.00 ATOM 541 CD1 ILE 34 1.685 3.924 −0.218 1.00 0.00 ATOM 542 HD11 ILE 34 1.227 2.947 −0.158 1.00 0.00 ATOM 543 HD12 ILE 34 1.922 4.272 0.778 1.00 0.00 ATOM 544 HD13 ILE 34 2.592 3.862 −0.801 1.00 0.00 ATOM 545 C ILE 34 −0.743 7.490 −0.992 1.00 0.00 ATOM 546 O ILE 34 −1.738 6.797 −1.017 1.00 0.00 ATOM 547 N VAL 35 −0.644 8.509 −0.177 1.00 0.00 ATOM 548 HN VAL 35 0.175 9.048 −0.161 1.00 0.00 ATOM 549 CA VAL 35 −1.773 8.837 0.750 1.00 0.00 ATOM 550 HA VAL 35 −2.296 7.928 1.006 1.00 0.00 ATOM 551 CB VAL 35 −1.216 9.472 2.028 1.00 0.00 ATOM 552 HB VAL 35 −0.771 10.427 1.791 1.00 0.00 ATOM 553 CG1 VAL 35 −2.352 9.673 3.034 1.00 0.00 ATOM 554 HG11 VAL 35 −3.111 8.923 2.874 1.00 0.00 ATOM 555 HG12 VAL 35 −2.781 10.655 2.900 1.00 0.00 ATOM 556 HG13 VAL 35 −1.964 9.584 4.037 1.00 0.00 ATOM 557 CG2 VAL 35 −0.158 8.546 2.637 1.00 0.00 ATOM 558 HG21 VAL 35 −0.220 8.585 3.714 1.00 0.00 ATOM 559 HG22 VAL 35 0.823 8.866 2.323 1.00 0.00 ATOM 560 HG23 VAL 35 −0.329 7.533 2.305 1.00 0.00 ATOM 561 C VAL 35 −2.751 9.808 0.080 1.00 0.00 ATOM 562 O VAL 35 −3.919 9.847 0.420 1.00 0.00 ATOM 563 N GLU 36 −2.289 10.591 −0.858 1.00 0.00 ATOM 564 HN GLU 36 −1.341 10.547 −1.110 1.00 0.00 ATOM 565 CA GLU 36 −3.195 11.563 −1.539 1.00 0.00 ATOM 566 HA GLU 36 −3.896 11.962 −0.820 1.00 0.00 ATOM 567 CB GLU 36 −2.362 12.709 −2.121 1.00 0.00 ATOM 568 HB1 GLU 36 −2.130 12.494 −3.154 1.00 0.00 ATOM 569 HB2 GLU 36 −1.442 12.803 −1.560 1.00 0.00 ATOM 570 CG GLU 36 −3.148 14.022 −2.036 1.00 0.00 ATOM 571 HG1 GLU 36 −2.483 14.820 −1.745 1.00 0.00 ATOM 572 HG2 GLU 36 −3.935 13.924 −1.300 1.00 0.00 ATOM 573 CD GLU 36 −3.764 14.346 −3.399 1.00 0.00 ATOM 574 OE1 GLU 36 −3.838 15.520 −3.730 1.00 0.00 ATOM 575 OE2 GLU 36 −4.152 13.417 −4.088 1.00 0.00 ATOM 576 C GLU 36 −3.962 10.866 −2.667 1.00 0.00 ATOM 577 O GLU 36 −5.076 11.237 −2.987 1.00 0.00 ATOM 578 N GLN 37 −3.372 9.874 −3.281 1.00 0.00 ATOM 579 HN GLN 37 −2.470 9.602 −3.012 1.00 0.00 ATOM 580 CA GLN 37 −4.059 9.163 −4.402 1.00 0.00 ATOM 581 HA GLN 37 −4.738 9.848 −4.893 1.00 0.00 ATOM 582 CB GLN 37 −3.018 8.680 −5.413 1.00 0.00 ATOM 583 HB1 GLN 37 −2.589 7.751 −5.071 1.00 0.00 ATOM 584 HB2 GLN 37 −2.239 9.423 −5.511 1.00 0.00 ATOM 585 CG GLN 37 −3.690 8.460 −6.770 1.00 0.00 ATOM 586 HG1 GLN 37 −3.755 9.399 −7.298 1.00 0.00 ATOM 587 HG2 GLN 37 −4.683 8.063 −6.617 1.00 0.00 ATOM 588 CD GLN 37 −2.867 7.468 −7.592 1.00 0.00 ATOM 589 OE1 GLN 37 −2.395 7.794 −8.663 1.00 0.00 ATOM 590 NE2 GLN 37 −2.672 6.264 −7.133 1.00 0.00 ATOM 591 HE21 GLN 37 −3.052 6.001 −6.268 1.00 0.00 ATOM 592 HE22 GLN 37 −2.145 5.620 −7.652 1.00 0.00 ATOM 593 C GLN 37 −4.852 7.960 −3.883 1.00 0.00 ATOM 594 O GLN 37 −5.831 7.556 −4.480 1.00 0.00 ATOM 595 N CYS 38 −4.422 7.368 −2.797 1.00 0.00 ATOM 596 HN CYS 38 −3.618 7.697 −2.345 1.00 0.00 ATOM 597 CA CYS 38 −5.138 6.167 −2.265 1.00 0.00 ATOM 598 HA CYS 38 −5.589 5.631 −3.086 1.00 0.00 ATOM 599 HB1 CYS 38 −4.649 4.387 −1.174 1.00 0.00 ATON 600 HB2 CYS 38 −3.674 5.787 −0.749 1.00 0.00 ATOM 601 C CYS 38 −6.231 6.567 −1.272 1.00 0.00 ATOM 602 O CYS 38 −7.400 6.350 −1.513 1.00 0.00 ATOM 603 CB CYS 38 −4.136 5.252 −1.563 1.00 0.00 ATOM 604 SG CYS 38 −2.871 4.725 −2.746 1.00 0.00 ATOM 605 N CYS 39 −5.860 7.122 −0.147 1.00 0.00 ATOM 606 HN CYS 39 −4.906 7.264 0.032 1.00 0.00 ATOM 607 CA CYS 39 −6.874 7.508 0.889 1.00 0.00 ATOM 608 HA CYS 39 −7.297 6.610 1.316 1.00 0.00 ATOM 609 HB1 CYS 39 −5.793 9.221 1.592 1.00 0.00 ATOM 610 HB2 CYS 39 −5.368 7.724 2.406 1.00 0.00 ATOM 611 C CYS 39 −8.010 8.348 0.274 1.00 0.00 ATOM 612 O CYS 39 −9.169 8.146 0.589 1.00 0.00 ATOM 613 CB CYS 39 −6.181 8.305 2.000 1.00 0.00 ATOM 614 SG CYS 39 −7.365 8.682 3.324 1.00 0.00 ATOM 615 N THR 40 −7.696 9.288 −0.585 1.00 0.00 ATOM 616 HN THR 40 −6.757 9.440 −0.821 1.00 0.00 ATOM 617 CA THR 40 −8.770 10.135 −1.197 1.00 0.00 ATOM 618 HA THR 40 −9.290 10.671 −0.415 1.00 0.00 ATOM 619 CB THR 40 −8.143 11.138 −2.166 1.00 0.00 ATOM 620 HB THR 40 −8.918 11.747 −2.607 1.00 0.00 ATOM 621 OG1 THR 40 −7.452 10.438 −3.192 1.00 0.00 ATOM 622 HG1 THR 40 −6.826 9.844 −2.773 1.00 0.00 ATOM 623 CG2 THR 40 −7.168 12.038 −1.408 1.00 0.00 ATOM 624 HG21 THR 40 −6.743 12.762 −2.087 1.00 0.00 ATOM 625 HG22 THR 40 −6.380 11.434 −0.983 1.00 0.00 ATOM 626 HG23 THR 40 −7.695 12.552 −0.619 1.00 0.00 ATOM 627 C THR 40 −9.766 9.251 −1.954 1.00 0.00 ATOM 628 O THR 40 −10.959 9.491 −1.933 1.00 0.00 ATOM 629 N SER 41 −9.284 8.238 −2.620 1.00 0.00 ATOM 630 HN SER 41 −8.318 8.071 −2.619 1.00 0.00 ATOM 631 CA SER 41 −10.192 7.333 −3.385 1.00 0.00 ATOM 632 HA SER 41 −11.218 7.626 −3.211 1.00 0.00 ATOM 633 CB SER 41 −9.865 7.469 −4.877 1.00 0.00 ATOM 634 HB1 SER 41 −10.646 6.998 −5.460 1.00 0.00 ATOM 635 HB2 SER 41 −8.924 6.989 −5.088 1.00 0.00 ATOM 636 OG SER 41 −9.772 8.847 −5.212 1.00 0.00 ATOM 637 HG SER 41 −8.925 9.172 −4.895 1.00 0.00 ATOM 638 C SER 41 −9.981 5.884 −2.900 1.00 0.00 ATOM 639 O SER 41 −9.741 5.659 −1.728 1.00 0.00 ATOM 640 N ILE 42 −10.066 4.901 −3.772 1.00 0.00 ATOM 641 HN ILE 42 −10.263 5.091 −4.711 1.00 0.00 ATOM 642 CA ILE 42 −9.857 3.491 −3.335 1.00 0.00 ATOM 643 HA ILE 42 −9.606 3.477 −2.283 1.00 0.00 ATOM 644 CB ILE 42 −11.139 2.683 −3.562 1.00 0.00 ATOM 645 HB ILE 42 −11.390 2.690 −4.611 1.00 0.00 ATOM 646 CG1 ILE 42 −12.270 3.324 −2.756 1.00 0.00 ATOM 647 HG11 ILE 42 −12.357 4.365 −3.023 1.00 0.00 ATOM 648 HG12 ILE 42 −12.052 3.240 −1.701 1.00 0.00 ATOM 649 CG2 ILE 42 −10.933 1.237 −3.093 1.00 0.00 ATOM 650 HG21 ILE 42 −10.172 1.212 −2.326 1.00 0.00 ATOM 651 HG22 ILE 42 −10.622 0.628 −3.928 1.00 0.00 ATOM 652 HG23 ILE 42 −11.860 0.853 −2.694 1.00 0.00 ATOM 653 CD1 ILE 42 −13.589 2.614 −3.059 1.00 0.00 ATOM 654 HD11 ILE 42 −13.641 2.388 −4.112 1.00 0.00 ATOM 655 HD12 ILE 42 −14.413 3.255 −2.785 1.00 0.00 ATOM 656 HD13 ILE 42 −13.642 1.696 −2.490 1.00 0.00 ATOM 657 C ILE 42 −8.699 2.897 −4.139 1.00 0.00 ATOM 658 O ILE 42 −8.885 2.325 −5.197 1.00 0.00 ATOM 659 N CYS 43 −7.499 3.050 −3.643 1.00 0.00 ATOM 660 HN CYS 43 −7.387 3.527 −2.793 1.00 0.00 ATOM 661 CA CYS 43 −6.295 2.525 −4.354 1.00 0.00 ATOM 662 HA CYS 43 −6.180 3.043 −5.293 1.00 0.00 ATOM 663 HB1 CYS 43 −4.550 1.830 −3.290 1.00 0.00 ATOM 664 HB2 CYS 43 −5.378 3.183 −2.530 1.00 0.00 ATOM 665 C CYS 43 −6.442 1.022 −4.617 1.00 0.00 ATOM 666 O CYS 43 −7.100 0.312 −3.880 1.00 0.00 ATOM 667 CB CYS 43 −5.059 2.767 −3.471 1.00 0.00 ATOM 668 SG CYS 43 −3.918 3.924 −4.276 1.00 0.00 ATOM 669 N SER 44 −5.813 0.539 −5.657 1.00 0.00 ATOM 670 HN SER 44 −5.283 1.137 −6.225 1.00 0.00 ATOM 671 CA SER 44 −5.883 −0.912 −5.981 1.00 0.00 ATOM 672 HA SER 44 −6.568 −1.404 −5.305 1.00 0.00 ATOM 673 CB SER 44 −6.360 −1.092 −7.422 1.00 0.00 ATOM 674 HB1 SER 44 −7.440 −1.078 −7.445 1.00 0.00 ATOM 675 HB2 SER 44 −6.006 −2.033 −7.808 1.00 0.00 ATOM 676 OG SER 44 −5.843 −0.035 −8.221 1.00 0.00 ATOM 677 HG SER 44 −6.246 −0.095 −9.091 1.00 0.00 ATOM 678 C SER 44 −4.485 −1.511 −5.823 1.00 0.00 ATOM 679 O SER 44 −3.498 −0.798 −5.827 1.00 0.00 ATOM 680 N LEU 45 −4.387 −2.808 −5.682 1.00 0.00 ATOM 681 HN LEU 45 −5.194 −3.364 −5.681 1.00 0.00 ATOM 682 CA LEU 45 −3.045 −3.444 −5.521 1.00 0.00 ATOM 683 HA LEU 45 −2.560 −3.041 −4.644 1.00 0.00 ATOM 684 CB LEU 45 −3.206 −4.956 −5.361 1.00 0.00 ATOM 685 HB1 LEU 45 −3.320 −5.413 −6.332 1.00 0.00 ATOM 686 HB2 LEU 45 −4.079 −5.164 −4.759 1.00 0.00 ATOM 687 CG LEU 45 −1.965 −5.522 −4.677 1.00 0.00 ATOM 688 HG LEU 45 −1.079 −5.102 −5.132 1.00 0.00 ATOM 689 CD1 LEU 45 −1.995 −5.150 −3.193 1.00 0.00 ATOM 690 HD11 LEU 45 −2.974 −5.355 −2.791 1.00 0.00 ATOM 691 HD12 LEU 45 −1.774 −4.099 −3.081 1.00 0.00 ATOM 692 HD13 LEU 45 −1.257 −5.732 −2.660 1.00 0.00 ATOM 693 CD2 LEU 45 −1.947 −7.044 −4.825 1.00 0.00 ATOM 694 HD21 LEU 45 −2.936 −7.435 −4.638 1.00 0.00 ATOM 695 HD22 LEU 45 −1.252 −7.467 −4.114 1.00 0.00 ATOM 696 HD23 LEU 45 −1.640 −7.303 −5.827 1.00 0.00 ATOM 697 C LEU 45 −2.187 −3.154 −6.754 1.00 0.00 ATOM 698 O LEU 45 −0.976 −3.060 −6.671 1.00 0.00 ATOM 699 N TYR 46 −2.809 −3.008 −7.895 1.00 0.00 ATOM 700 HN TYR 46 −3.786 −3.087 −7.930 1.00 0.00 ATOM 701 CA TYR 46 −2.045 −2.721 −9.141 1.00 0.00 ATOM 702 HA TYR 46 −1.290 −3.477 −9.281 1.00 0.00 ATOM 703 CB TYR 46 −3.007 −2.732 −10.336 1.00 0.00 ATOM 704 HB1 TYR 46 −3.810 −2.033 −10.158 1.00 0.00 ATOM 705 HB2 TYR 46 −3.414 −3.724 −10.459 1.00 0.00 ATOM 706 CG TYR 46 −2.264 −2.335 −11.590 1.00 0.00 ATOM 707 CD1 TYR 46 −2.461 −1.062 −12.137 1.00 0.00 ATOM 708 HD1 TYR 46 −3.146 −0.369 −11.659 1.00 0.00 ATOM 709 CD2 TYR 46 −1.376 −3.233 −12.193 1.00 0.00 ATOM 710 HD2 TYR 46 −1.220 −4.211 −11.760 1.00 0.00 ATOM 711 CE1 TYR 46 −1.770 −0.686 −13.295 1.00 0.00 ATOM 712 HE1 TYR 46 −1.921 0.296 −13.719 1.00 0.00 ATOM 713 CE2 TYR 46 −0.683 −2.856 −13.349 1.00 0.00 ATOM 714 HE2 TYR 46 0.002 −3.547 −13.819 1.00 0.00 ATOM 715 CZ TYR 46 −0.882 −1.583 −13.902 1.00 0.00 ATOM 716 OH TYR 46 −0.199 −1.212 −15.042 1.00 0.00 ATOM 717 HH TYR 46 0.494 −0.600 −14.786 1.00 0.00 ATOM 718 C TYR 46 −1.371 −1.349 −9.025 1.00 0.00 ATOM 719 O TYR 46 −0.223 −1.183 −9.391 1.00 0.00 ATOM 720 N GLN 47 −2.080 −0.369 −8.541 1.00 0.00 ATOM 721 HN GLN 47 −3.008 −0.524 −8.266 1.00 0.00 ATOM 722 CA GLN 47 −1.485 0.990 −8.416 1.00 0.00 ATOM 723 HA GLN 47 −1.092 1.295 −9.376 1.00 0.00 ATOM 724 CB GLN 47 −2.567 1.969 −7.974 1.00 0.00 ATOM 725 HB1 GLN 47 −2.118 2.917 −7.729 1.00 0.00 ATOM 726 HB2 GLN 47 −3.075 1.572 −7.110 1.00 0.00 ATOM 727 CG GLN 47 −3.573 2.161 −9.110 1.00 0.00 ATOM 728 HG1 GLN 47 −4.325 1.388 −9.057 1.00 0.00 ATOM 729 HG2 GLN 47 −3.060 2.100 −10.060 1.00 0.00 ATOM 730 CD GLN 47 −4.246 3.531 −8.980 1.00 0.00 ATOM 731 OE1 GLN 47 −4.262 4.117 −7.915 1.00 0.00 ATOM 732 NE2 GLN 47 −4.809 4.068 −10.028 1.00 0.00 ATOM 733 HE21 GLN 47 −4.797 3.595 −10.887 1.00 0.00 ATOM 734 HE22 GLN 47 −5.241 4.943 −9.957 1.00 0.00 ATOM 735 C GLN 47 −0.346 0.951 −7.393 1.00 0.00 ATOM 736 O GLN 47 0.610 1.696 −7.495 1.00 0.00 ATOM 737 N LEU 48 −0.436 0.081 −6.417 1.00 0.00 ATOM 738 HN LEU 48 −1.212 −0.516 −6.363 1.00 0.00 ATOM 739 CA LEU 48 0.651 −0.016 −5.396 1.00 0.00 ATOM 740 HA LEU 48 1.124 0.947 −5.295 1.00 0.00 ATOM 741 CB LEU 48 0.065 −0.441 −4.042 1.00 0.00 ATOM 742 HB1 LEU 48 0.833 −0.930 −3.460 1.00 0.00 ATOM 743 HB2 LEU 48 −0.748 −1.132 −4.208 1.00 0.00 ATOM 744 CG LEU 48 −0.457 0.777 −3.266 1.00 0.00 ATOM 745 HG LEU 48 −1.261 1.240 −3.821 1.00 0.00 ATOM 746 CD1 LEU 48 −0.977 0.309 −1.910 1.00 0.00 ATOM 747 HD1 LEU 48 −2.009 0.009 −2.004 1.00 0.00 ATOM 748 HD12 LEU 48 −0.896 1.117 −1.199 1.00 0.00 ATOM 749 HD13 LEU 48 −0.385 −0.528 −1.572 1.00 0.00 ATOM 750 CD2 LEU 48 0.667 1.794 −3.037 1.00 0.00 ATOM 751 HD21 LEU 48 1.622 1.292 −3.091 1.00 0.00 ATOM 752 HD22 LEU 48 0.555 2.248 −2.064 1.00 0.00 ATOM 753 HD23 LEU 48 0.617 2.558 −3.797 1.00 .0.00 ATOM 754 C LEU 48 1.707 −1.051 −5.829 1.00 0.00 ATOM 755 O LEU 48 2.726 −1.202 −5.182 1.00 0.00 ATOM 756 N GLU 49 1.473 −1.775 −6.905 1.00 0.00 ATOM 757 HN GLU 49 0.646 −1.650 −7.410 1.00 0.00 ATOM 758 CA GLU 49 2.467 −2.800 −7.360 1.00 0.00 ATOM 759 HA GLU 49 2.543 −3.576 −6.616 1.00 0.00 ATOM 760 CB GLU 49 2.004 −3.420 −8.684 1.00 0.00 ATOM 761 HB1 GLU 49 2.865 −3.647 −9.297 1.00 0.00 ATOM 762 HB2 GLU 49 1.371 −2.717 −9.205 1.00 0.00 ATOM 763 CG GLU 49 1.215 −4.711 −8.413 1.00 0.00 ATOM 764 HG1 GLU 49 0.227 −4.613 −8.820 1.00 0.00 ATOM 765 HG2 GLU 49 1.144 −4.879 −7.351 1.00 0.00 ATOM 766 CD GLU 49 1.916 −5.902 −9.074 1.00 0.00 ATOM 767 OE1 GLU 49 2.492 −6.701 −8.351 1.00 0.00 ATOM 768 OE2 GLU 49 1.866 −5.996 −10.289 1.00 0.00 ATOM 769 C GLU 49 3.844 −2.156 −7.574 1.00 0.00 ATOM 770 O GLU 49 4.860 −2.823 −7.537 1.00 0.00 ATOM 771 N ASN 50 3.881 −0.869 −7.823 1.00 0.00 ATOM 772 HN ASN 50 3.046 −0.357 −7.869 1.00 0.00 ATOM 773 CA ASN 50 5.185 −0.185 −8.071 1.00 0.00 ATOM 774 HA ASN 50 5.893 −0.902 −8.457 1.00 0.00 ATOM 775 CB ASN 50 4.975 0.920 −9.111 1.00 0.00 ATOM 776 HB1 ASN 50 5.648 1.741 −8.908 1.00 0.00 ATOM 777 HB2 ASN 50 3.953 1.271 −9.062 1.00 0.00 ATOM 778 CG ASN 50 5.258 0.368 −10.509 1.00 0.00 ATOM 779 OD1 ASN 50 6.361 0.475 −11.005 1.00 0.00 ATOM 780 ND2 ASN 50 4.300 −0.219 −11.169 1.00 0.00 ATOM 781 HD21 ASN 50 3.409 −0.305 −10.770 1.00 0.00 ATOM 782 HD22 ASN 50 4.469 −0.577 −12.066 1.00 0.00 ATOM 783 C ASN 50 5.743 0.428 −6.778 1.00 0.00 ATOM 784 O ASN 50 6.350 1.482 −6.806 1.00 0.00 ATOM 785 N TYR 51 5.553 −0.216 −5.650 1.00 0.00 ATOM 786 HN TYR 51 5.063 −1.063 −5.643 1.00 0.00 ATOM 787 CA TYR 51 6.088 0.349 −4.372 1.00 0.00 ATOM 788 HA TYR 51 6.472 1.335 −4.569 1.00 0.00 ATOM 789 CB TYR 51 4.956 0.454 −3.351 1.00 0.00 ATOM 790 HB1 TYR 51 5.360 0.475 −2.351 1.00 0.00 ATOM 791 HB2 TYR 51 4.289 −0.387 −3.464 1.00 0.00 ATOM 792 CG TYR 51 4.204 1.725 −3.623 1.00 0.00 ATOM 793 CD1 TYR 51 3.397 1.803 −4.753 1.00 0.00 ATOM 794 HD1 TYR 51 3.300 0.945 −5.398 1.00 0.00 ATOM 795 CD2 TYR 51 4.331 2.827 −2.772 1.00 0.00 ATOM 796 HD2 TYR 51 4.953 2.763 −1.891 1.00 0.00 ATOM 797 CE1 TYR 51 2.709 2.982 −5.046 1.00 0.00 ATOM 798 HE1 TYR 51 2.086 3.038 −5.925 1.00 0.00 ATOM 799 CE2 TYR 51 3.641 4.011 −3.057 1.00 0.00 ATOM 800 HE2 TYR 51 3.738 4.862 −2.402 1.00 0.00 ATOM 801 CZ TYR 51 2.830 4.090 −4.197 1.00 0.00 ATOM 802 OH TYR 51 2.152 5.257 −4.482 1.00 0.00 ATOM 803 HH TYR 51 1.390 5.308 −3.900 1.00 0.00 ATOM 804 C TYR 51 7.230 −0.518 −3.818 1.00 0.00 ATOM 805 O TYR 51 7.923 −0.117 −2.901 1.00 0.00 ATOM 806 N CYS 52 7.442 −1.687 −4.368 1.00 0.00 ATOM 807 HN CYS 52 6.879 −1.991 −5.103 1.00 0.00 ATOM 808 CA CYS 52 8.546 −2.564 −3.875 1.00 0.00 ATOM 809 HA CYS 52 8.538 −2.580 −2.794 1.00 0.00 ATOM 810 HB1 CYS 52 9.063 −4.649 −3.923 1.00 0.00 ATOM 811 HB2 CYS 52 8.548 −3.997 −5.475 1.00 0.00 ATOM 812 C CYS 52 9.886 −2.011 −4.367 1.00 0.00 ATOM 813 O CYS 52 10.029 −1.645 −5.519 1.00 0.00 ATOM 814 CB CYS 52 8.362 −3.989 −4.411 1.00 0.00 ATOM 815 SG CYS 52 6.671 −4.570 −4.093 1.00 0.00 ATOM 816 N ASN 53 10.868 −1.949 −3.501 1.00 0.00 ATOM 817 HN ASN 53 10.726 −2.252 −2.580 1.00 0.00 ATOM 818 CA ASN 53 12.203 −1.421 −3.913 1.00 0.00 ATOM 819 HA ASN 53 12.072 −0.513 −4.483 1.00 0.00 ATOM 820 CB ASN 53 13.041 −1.123 −2.665 1.00 0.00 ATOM 821 HB1 ASN 53 13.670 −1.974 −2.443 1.00 0.00 ATOM 822 HB2 ASN 53 12.384 −0.933 −1.828 1.00 0.00 ATOM 823 CG ASN 53 13.918 0.105 −2.919 1.00 0.00 ATOM 824 OD1 ASN 53 15.130 0.023 −2.861 1.00 0.00 ATOM 825 ND2 ASN 53 13.354 1.248 −3.198 1.00 0.00 ATOM 826 HD21 ASN 53 12.378 1.316 −3.245 1.00 0.00 ATOM 827 HD22 ASN 53 13.908 2.041 −3.361 1.00 0.00 ATOM 828 C ASN 53 12.920 −2.464 −4.773 1.00 0.00 ATOM 829 OT1 ASN 53 13.826 −2.084 −5.497 1.00 0.00 ATOM 830 OT2 ASN 53 12.553 −3.625 −4.690 1.00 0.00 END

Example 3 Relative Folding Stability of Asp^(B28)IP Analogs

For evaluation of folding stability for insulin precursor analogs, denaturation samples were prepared by combining different ratios of insulin precursor analog and GuHCl stock solutions with 10 mM Tris/ClO₄ ⁻, pH 8.0. Protein stock solutions were typically 0.06 mM in 10 mM Tris/ClO₄ ⁻, pH 8.0. GuHCl stock solutions were 8.25 M in 10 mM Tris/CIO₄ ⁻, pH 8.0. CD spectra were recorded with a Jasco J-715 Spectropolarimeter calibrated with (+)-10-camphorsulfonic acid. All spectra were recorded at 20° C. The denaturation samples were scanned from 250 to 218 nm. Typical cell path length and protein concentration were 0.5 cm and 3 μM, respectively. All spectra were smoothed before subtraction of appropriate solvent blanks. The circular dichroism is expressed as, AE, based on the molar concentration of peptide bond. For presentation purpose each curve is normalized to a 0–1 scale by dividing the observed change at each point by the total change observed in the experiment.

Data analysis. GuHCl denaturation curves were analyzed by assuming that the folding/unfolding transition is two-state as described by Santoro & Bolen (1988) Biochemistry 0.27:8063–8068 and Kaarsholm et al. (1993) Biochemistry 32:10773–10778, both of which publications are specifically incorporated herein by reference for teaching methods of calculating stability by GuHCl denaturation. This analysis yields a number of parameters including the GuHCl concentration at the midpoint of the denaturation curve, Cmid reflecting the concentration of denaturant necessary to unfold one-half of the protein population. An increase in folding stability is thus manifest by an increased value of Cmid. Equilibrium constants can be obtained at each denaturant concentration using K=(Δε_(N)−Δε)/(Δε_(U)), where Δε is the observed value of the CD, and Δε_(N) and Δε_(U) represent the CD values for native and unfolded forms, respectively, at the given GuHCl concentration (Pace, 1975). Values for Δε_(N) and Δε_(U) at GuHCl concentrations in the transition region are obtained by linear extrapolation of the pre- and post-transition baselines into the transition region, i.e. Δε_(N)=Δε⁰ _(N)+m_(N)[GuHCl], and Δε_(U)=Δε⁰ _(U)+m_(U)[GuHCl], where Δε⁰ _(N) and Δε⁰ _(U) are intercepts, and m_(N) and m_(U) are slopes of the pre- and post-transition baselines, respectively. The free energy of unfolding at a given denaturant concentration in the transition zone is given by ΔG=−RTlnK. Assuming a linear dependence of ΔG on denaturant concentration: ΔG=ΔG_(H) ₂ _(O)−m[GuHCl], where ΔG_(H) ₂ _(O) is the value of ΔG in the absence of denaturant, and m is a measure of the dependence of ΔG on denaturant concentration. Hence, ΔG values derived from K in the transition zone may be extrapolated back to 0 M denaturant to give ΔG ₂ _(O). The relationship between Δε and [GuHCl] for the complete unfolding curve is shown in Eq. 1 (Santoro & Bolen, 1988):

$\begin{matrix} {{\Delta ɛ} = \frac{\left( {{\Delta ɛ}_{N}^{0} + {m_{N}\left\lbrack {{Gu}{HCl}} \right\rbrack}} \right) + {\left( {{\Delta ɛ}_{U}^{0} + {m_{U}\left\lbrack {{Gu}{HCl}} \right\rbrack}} \right){\exp\left( {{- \left( {{\Delta\; G_{H_{2}0}} - {m\left\lbrack {{Gu}{HCl}} \right\rbrack}} \right)}/{RT}} \right)}}}{1 + {\exp\left( {{- \left( {{\Delta\; G_{H_{2}0}} - {m\left\lbrack {{Gu}{HCl}} \right\rbrack}} \right)}/{RT}} \right)}}} & (1) \end{matrix}$ With Δε as the response and [GuHCl]as the independent variable, eq. (1) is subject to nonlinear least squares analysis using the NLIN procedure of PC SAS (SAS Inc. Cary, N.C.). Six parameters then describe the denaturation curve: Δε⁰ _(N), Δε⁰ _(U), m_(N), m_(U), m, and ΔG_(H) ₂ _(O). In addition, the GuHCl concentration at the midpoint of the denaturation curve, Cmid, is given by ΔG_(H) ₂ _(O)/m.

Evaluation of the relative folding stability of Asp^(B28)IP derivative molecules with C-peptide Met Trp Lys (Asp^(B28)IP(MetTrpLys)) was evaluated relative to Asp^(B28)IP. The results show that the Asp^(B28)IP(MetTrpLys) molecule was much more stable than Asp^(B28)IP (FIG. 5), as evidenced by the change in Cmid. While Cmid for Asp^(B28)IP is approximately about 5.5 M GuHCl, that of Asp^(B28)IP(MetTrpLys) is increased to at least about 6.5 M GuHCl, an increase of approximately 18%.

Example 4

The insulin analogue precursor Asp^(B28)IP(EWK) was produced culturing yeast strain MT663 transformed with an expression plasmid expressing a YAP3-TA39-EEGEPK(SEQ ID NO:17)-Asp^(B28)IP(EWK) fusion protein or a YAP3-TA57-EEGEPK(SEQ ID NO:17)-Asp^(B28)IP(EWK) fusion protein.

cDNA encoding the leader sequences YAP3-TA39 and YAP3-TA57 and cDNA encoding the Asp^(B28)IP(EWK) and the N-terminal extension were cloned into an expression vector of the C-POT type using standard techniques (Sambrook J, Fritsch EF and Maniatis T, Molecular cloning, Cold spring Harbour laboratory press, 1989). The DNA and inferred amino acids sequences are shown in FIGS. 8 and 9.

Table 6 shows the yields. Fermentation was conducted at 30° C. for 72 h in 5 ml YPD. IP yield was determined by RP-HPLC of the culture supernatant and is expressed relative to the IP yield of a control strain.

In Table 6, “α*” indicates an α-factor leader in which the C-terminus up to the LysArg has been modified from “SLD (SerLeuAsp)” to “SMA (SerMetAla)” and “ex4” is an N-terminal extension with the amino acid sequence EEAEAEAPK(SEQ ID NO:4). YAP3 is the YAP3 signal sequence. TA39 is a synthetic pro-sequence QPIDDTESNTTSVNLMADDTESRFATNTTLAGGLDVVNLISMAKR (SEQ ID NO:16). The sequence EEGEPK(SEQ ID NO:17) is an N-terminal extension to the B-chain of the insulin analogue. TA57 is a synthetic pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR (SEQ ID NO: 18).

TABLE 6 Leader Precursor N-terminal extension C-peptide Yield* a*-ex4 Asp^(B28)IP GluGluAlaGluAlaGluAlaProLys None 100   (SEQ ID NO: 4) YAP3-TA39 Asp^(B28)IP GluGluGlyGluProLys GluTrpLys 531% (SEQ ID NO: 17) YAP3-TA57 Asp^(B28)IP GluGluGlyGluProLys GluTrpLys 500% (SEQ ID NO: 17) 

1. An insulin-Asp^(B28) precurser comprising a connecting peptide (C-peptide) being cleavable from the A and B chains, said connecting peptide comprising one aromatic amino acid residue which is less than 5 Å away from at least one of the residues in position B11, B12, or B26 in the B-chain.
 2. A precursor according to claim 1, wherein the connecting peptide is of up to 15 amino acid residues in length.
 3. A precursor according to claim 1, wherein the connecting peptide is of up to 9 amino acid residues.
 4. A precursor according to claim 1, wherein the connecting peptide is of up to 5 amino acid residues.
 5. A precursor according to claim 1, wherein the connecting peptide is of up to 3 amino acid residues.
 6. A precursor according to claim 1, wherein the connecting peptide comprises up to 5 aromatic amino acid residues.
 7. A precursor according to claim 1, wherein the connecting peptide comprises one aromatic amino acid residue.
 8. A precursor according to claim 1, wherein the connecting peptide has a Lys or Arg immediately N-terminal to the A chain.
 9. A precursor according to claim 8, wherein one aromatic amino acid residue is immediately N-terminal to the Lys or Arg.
 10. A precursor according to claim 9, wherein the aromatic amino acid residues positioned immediately N-terminal to the Lys or Arg is less than 5 Å away from at least one of the residues in positions B11, B12, or B26 in the B-chain.
 11. A precursor according to claim 1, wherein the precursor exhibits an increased Cmid stability in solution relative to a precursor which does not comprise an aromatic amino acid residue in the connecting peptide.
 12. A precursor of claim 11, wherein Cmid is higher than about 5.5 M GuHCl.
 13. A precursor of claim 11, wherein Cmid is higher than about 6.0 M GuNCl.
 14. A precursor of claim 11, wherein Cmid is at least about 6.5 M GuLHCl.
 15. A process for making an insulin precursor or an insulin analog precursor, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or an insulin analog precursor according to claim 1 under suitable culture conditions for expression of said precursor; and (ii) isolating the expressed precursor.
 16. A process according to claim 15, wherein the host cell is a yeast host cell.
 17. A process for making insulin or an insulin analog, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or an insulin analog precursor according to claim 1 under suitable culture conditions for expression of said precursor; (ii) isolating the precursor from the culture medium and (iii) converting the precursor into insulin or an insulin analog by in vitro chemical or enzymatic conversion.
 18. A process according to claim 17, wherein the host cell is a yeast host cell. 